This study reports field and laboratory experiments comprising morphological and molecular characterization, genotype x environment interactions and yield analysis of top vs. bottom segments, in 16 sugarcane genotypes using morphological attributes and molecular techniques. These experiments were carried out during 2010-11 and 2011-12. Morphological characterization was undertaken at Sugar Crops Research Institute (SCRI), Mardan-Pakistan while molecular characterization was carried out at Plant Genetic Resource Program (PGRP), National Agricultural Research Center (NARC), Islamabad-Pakistan and Biotechnological Laboratory of the Southern Illinois University, Carbondale, USA. The genotype by location interaction trials were planted at two different locations of the Khyber Pakhtunkhwa province of Pakistan. The study of top vs. bottom segments performance was conducted on farmer’s field at Dargai (Malakand). All these experiments were conducted in triplicate RCB, Design with plot area of 67m2 (10m x 6.7m). The cluster analysis based on 20 morphological attributes divided 16 sugarcane genotypes into four different clusters by UPGMA method at Euclidean distance of 4.99. Pair wise Euclidean distance ranged from 2.30 to 7.43. The highest Euclidean distance was recorded between genotypes Hoth127 and MS91CP238 while the lowest was recorded between genotypes S96SP1215 and MS99HO93. The cluster-I comprised four genotypes. Common traits in cluster-I genotypes were medium cane height and leaf shape and no streaks and pubescence. The cluster- II had only one sugarcane genotype with attributes intermediate among other clusters. Cluster-III was composed of seven genotypes and having attributes intermediate among other clusters. Cluster-IV had four genotypes which commonly possessed small legule size and light red dewlap color. It was concluded that morphological traits evaluated in this study could be used for varietal identification and selection of desirable genotypes in sugarcane. Forty six microsatellite (SSR) markers were utilized to detect genetic diversity on molecular basis among 16 genotypes of sugarcane. The results showed considerable level of genetic diversity among the material used. Out of the 164 loci, 71.34% were polymorphic while 28.66% were monomorphic with an average of 3.57 alleles per locus of SSR. Of these 46 primers, only 13 (28.26%) produced polymorphic bands, 10 (21.74%) produced monomorphic and 23 (50%) produced both polymorphic and monomophic. The highest number of bands (10) were generated by two SSR primers (SCM16 and UGSM574) while 11 primers (SMC336BS, MCSA053C10, SOMS118, SMC1751CL, mSSCIR3, SMC7CUQ, SMC1604SA, SMC851MS, SOMS156, UGSM154 and UGSM312) produced one band. The PIC value of the polymorphic loci in 16 sugarcane genotypes ranged from 0.009 to 0.947 with an average of 0.490 per locus. The average number of alleles per locus were 3.57, whereas the average number of alleles per polymorphic locus were 3.30. The dendrogram grouped the 16 promising sugarcane genotypes vii into four main clusters. The cluster-I comprised two genotypes whereas cluster-II possessed five genotypes. The genotype MS92CP979 was separately grouped into cluster-III. The cluster-IV consisted of eight genotypes. The similarity matrix showed pair wise genetic similarity range from 71% to 93%. The highest genetic similarity (93%) was detected between genotypes MS99HO391 and S97CP288 whereas the lowest genetic similarity of 71% was detected between MS94CP15 and CP89831. In both (morphological and molecular) characterization study, cluster analysis classified 16 sugarcane genotypes into four main groups at Euclidean distance of 4.99 and 82.50% coefficient of similarity, respectively. The comparison of both types dendrogram illustrated that some genotypes were grouped into same cluster while the rest into different. In both studies genotype MS91CP272 was categorized into cluster-I while genotype MS99HO391 was into cluster-II. Similarly, genotypes MS99HO388, CP77400, Hoth127 and MS99HO388 were grouped into cluster-IV in both dendrograms. Both morphological and molecular markers were highly effective in assessment of genetic diversity and genotype identification in sugarcane. Combined analysis of variance was used to identify the presence of genotype x location interactions from replicated multi-environment trials. The data on nine quantitative and four qualitative parameters revealed highly significant variations among the genotypes for germination %, tillers, plant height, nodes plant-1, internode length and cane yield at 1% probability level. It was observed that none of the genotypes could show superiority with respect to all attributes. However, the mean performance over locations and years indicated that the check genotype Mardan93 remained superior by showing maximum buds germination of 42.26%. The maximum number of tillers (259.17), plant height (189.41cm), nodes plant-1 (22.23), internode length (16.97cm), cane diameter (23.29mm), cane yield (68.42 t ha-1), millable canes (87.83), c.brix (20.07%), pol (17.06%), purity (86.20%), sugar recovery (10.57%) and sugar yield (7.07 t ha-1) was recorded for genotypes MS91CP238, MS99HO391, Hoth127, MS94CP15, S97CP288, MS99HO317, MS92CP979, MS91CP272, MS99HO93, MS99HO93, MS99HO93 and MS99HO317, respectively against check genotypes. It was concluded that genotypes MS99HO317, MS99HO93, MS92CP979 and MS91CP238 were superior at SCRI, Mardan on the basis of tillering ability, milliable canes, cane yield, sugar recovery and sugar yield. At test location-II, the cultivars MS91CP272, MS99HO391, MS94CP15 and MS99HO391 were superior on the basis of tillers, milliable canes, sugar recovery and sugar yield compared to other genotypes. On the basis of combined over years and locations performance, genotypes MS99HO317, MS91CP238, MS92CP979 and CP89831 performed best for germination %, tillers, number of millable canes, cane yield, sugar yield and sugar recovery. Combined ANOVA over years and segments revealed significant differences among sugarcane genotypes for germination %, tillers, plant height, nodes plant-1, internode length, cane yield, millable canes and sugar yield. Highly significant differences were observed for segment x genotype interactions in cane yield and sugar yield and showed significant (p≤0.05) differences for millable canes only. Mean results showed that highest germination (65.92% and 47.58%) and tillers (164.95 and 137.00) in top and bottom segments, respectively were exhibited by genotype MS91CP238 against the check genotypes. Maximum plant height (233.64 and 234.17cm) in top and bottom segments was recorded for genotypes Hoth127 and MS94CP15, respectively. Maximum nodes plant-1 (20.22, 17.78) in top and bottom segments were recorded for genotypes Hoth127 and MS99HO388, respectively. Maximum internode length (16.45 and 17.04cm) in top and bottom segments was recorded for genotypes MS92CP979 and MS94CP15, respectively. Maximum cane diameter (23.55 and 23.00 mm) in top and bottom segments was recorded for genotypes S96SP1215 and MS99HO388. The highest cane yield (59.72 and 64.10 t ha-1) and milliable canes (127.26 and 95.81) in top and bottom segments were recorded for genotypes MS99HO388 and MS91CP238, respectively. The highest c. brix (19.35 and 21.12%) and pol viii (18.10 and 16.85%) in top and bottom segments were recorded for genotypes MS99HO93 and CP89831, respectively. The highest purity (94.32 and 85.55 %) in top and bottom segments was recorded for genotype MS91CP238 and MS94CP15, respectively. The maximum sugar recovery (12.23 and 10.49%) in top and bottom segments was recorded for genotypes MS99HO93 and S97CP288, respectively. The highest sugar yield (7.05 and 5.96 t ha-1) in top and bottom segments was recorded for genotypes MS99HO388 and MS91CP238, respectively. On the basis of overall combined over years performance, it was concluded that genotypes MS99HO388, Hoth127, S96SP1215, MS91CP238 and MS99HO388 showed better performance in top segments regarding germination, tillers, plant height, nodes plant-1, cane diameter, cane yield, millable canes, c.brix, pol, purity, sugar recovery and sugar yield. Genotypes MS94CP15, MS99HO388, MS91CP238 and CP89831 displayed better performance in bottom segments regarding germination, tillers, plant height, nodes plant-1, internode length, cane diameter, cane yield, millable canes, c.brix, purity, sugar recovery and sugar yield. There were some genotypes MS91CP238, MS99HO388, MS94CP15 and Hoth127 that proved to be superior in both top and bottom segments regarding parameters germination, tillers, plant height, nodes plant-1, internode length, cane diameter, cane yield, millable canes, c.brix, purity, sugar recovery and sugar yield. Overall this study revealed that both morphological and molecular characterization are useful techniques for detection of diversity and best genotype/varietal identification and selection. The genotype x environment interactions and top vs. bottom segments performance highlighted the best genotypes on the basis of yield and growth parameters. These genotypes could be used in future sugarcane breeding programs to develop high yielding sugarcane varieties." xml:lang="en_US