Tomato (Solanum lycopersicum L.) is most nutritionally and economically important crop in Pakistan and around the world. Early blight (EB) in one of tomato dreadful disease caused by fungal pathogen Alternaria solani (Ellis and Martin, 1882) causes major yield losses. Prolonged humidity due to extensive rain and warm conditions during growing season of tomato make this disease unmanageable through sanitation, crop rotation, fungicide application etc. leaving cultivation of resistant varieties as the most appropriate control measure. This study focused mainly to screen phenotypic and molecular marker assisted R gene analogues against A. solani for EB in tomato • Initially twenty nine tomato germlines/varieties were evaluated for their resistance against A. solani by artificially inoculating 15-days old tomato seedlings grown in potted soil in green house. Among them, 8 germlines/varieties were grouped as resistant (RR), seven as moderately resistant (MR); six as moderately susceptible (MS) and eight as susceptible (SS) on the basis of percent disease index (PDI) and growth inhibition index (GII) at 30 DAI (days after inoculation) and 60 DAI. Physiological parameters i.e. total chlorophyll content and carotenoids decreased in all inoculated germlines/varieties with most significant reduction in moderately susceptible and susceptible germlines/varieties at both 30 DAI and 60 DAI. However, biochemical attributes i.e. total phenolics, total protein content and activities of antioxidant enzymes [(peroxidase (POX), polyphenol oxidase (PPO), phenylalanine ammonia lyase (PAL) and catalase (CAT)] increased in all inoculated germlines/varieties with respect to corresponding control. Total phenolics, total protein content, POX, PPO and PAL activities were higher in susceptible than in resistant germlines/varieties. Whereas, activity of CAT was the highest in resistant and least in susceptible groups. • RAPD assay using 50 RAPD decamers reveled polymorphism with 32 decamers. The polymorphic pattern acquired by 32 RAPD decamers produced 181 loci (5.7 loci per primer), 157 were polymorphic (86%) and 24 were monomorphic (14%) in all 29 germlines/varieties. Marker A-04, A-10, B-05, L-15, L-17, M-04 and M-07 produced bands ranges from 500-1031 bp specifically in RR group and M-08 and M-10 generated loci (500-800 bp) exclusively in MR. Similarly, A-18, L-06, L-09, L-11, M-09 and OPJ- 10 produced bands from 200-1500 bp in MS and L-09 amplified the loci of 1200 bp only in completely SS. The cluster analysis revealed 60% homology among all germlines/varieties and segregated them into two major groups. Group I was further divided into 3 sub-groups including RR (575, Zeba, KHT-105, KHT-106-G, Advanta- 1206, Mishal, Namadar and Savana); MR (Advanta-1225, Baby red, Yaqoot, Maharaja, Commander, KHT-101 B and TO-1057) and MS (FMC-339, Shangrilla, Thorgal, Rio Grande, Roma and Surkhab). Group 2 comprised of SS germlines (AS-2565, Cluster- 809, GHT-1, Naqeeb, GHT-2, Mongal, Cluster-805 and Roshan). • Resistance gene analogues (RGAs) screening was conducted with 12 tomato germlines/varieties i.e. 4 from RR, 3 from MR, 3 from MS and 2 from SS categorized through PDI, GII and RAPD analysis. DNA of selected 12 germlines/varieties was amplified with 10 RGA primers pairs from conserved region of leucine-rich repeat (LRR), nucleotide binding site (NBS) and protein kinase (Ptokin) domains. Only 3 RGAs xv primer pairs i.e. PtoFen (S+AS), Ptokin (3+4) and Ptokin (1+2) successfully generated discrete PCR products ranges from 200-1100 bp. Primer pair PtoFen (S+AS) produced bands in all germlines/varieties, Band of 533 bp was amplified only in SS and in one variety (Roma) in MS, while band of 511 bp was amplified in the remaining germlines/varieties. The sequence of PtoFen RGAs’ from RR, MR and MS showed the maximum homology of 97-100% with serine/threonine protein kinase protein and had Pkc domain encoding region at 121 to 510 nucleotide. Primers Ptokin 3 and Ptokin 4 generated PCR product of ≃130 bp in SS, while band of ≃208 bp was produced in other germlines/varieties. Moreover, only the band sequenced from RR, MR and MS showed homology of 97-99% with Lycopersicum hirsutum clone RGA sequences. Primers Ptokin (1+2) produced two discrete bands of ≃ 1 kb and 1.3 kb only in RR, MR and MS germlines/varieties. No band was generated in SS germlines. However, sequence and cluster analysis dichotomize the bands of ≃ 1 kb in 2 MR + 2 MS (TO-1057, Yaqoot, Surkhab, FMC-339) and≃ 1.3 kb in 3 RR (Advanta-1209, Zeba and 575) into two divisions with 46% homology and 0.26 genetic distance. It was concluded that combination of phenotypic and genotypic (RGAs) approaches with bioinformatics tools could be used to identify EB resistance in tomato. This study would be a guideline towards solution to devastating fungal pathogen through developing resistant cultivars that could be later used in breeding program for sustainable crop production.