Cultivatedtomato(Solanumlycopersicum)isreportedwithlowlevelsof genetic diversity for biotic stresses. Tomato crop meets substantial damages due to several pathogens causing serious diseases. Fungicide application and cultural practices were used to manage the disease previously. The factors includinglimitedavailabilityofcommerciallyacceptableresistantcultivar and negative environmental impact of chemical application have prompted the use of identification and use of genetic resistance to complement other practices to manage the disease. The current study was performed to determine the extent of genetic variation among the selected tomato germplasm for biotic stress and incorporation of required genetic diversity into elite lines. Germplasm comprised of 46 genotypes including cultivated varieties and advanced lines developed at Hazara Agricultural Research Station, Abbottabad, Pakistan were used in this study. Initially, 46 genotypes were screened for resistant genes for fusarium wilt (I1), late blight (Ph3), bacterial wilt (Bwr-12) and tomato yellow leaf curl virus (Ty-2). Identification of resistant gene was done using SSR markers that identified 17 genotypes bearing I1 gene conferring resistance to Fusarium wilt. SCAR marker was used to screen late blight resistance gene (Ph-3). Total of 13 genotypes were identified for Ph-3 gene. 16 genotypes were identified for Bwr-12 gene conferring resistance to bacterial wilt while 12 genotypes were identified for Ty-2 gene resistant to tomato yellow leaf curl virus. Gene pyramiding for disease resistant genes was carried out by conventional crossing of cultivated variety (Roma) and advanced line (1008) . Each of the linebeartworequiredgenesandmarkerassistedselectionwasusedto identify the target segregants until the identification of stable lines with multiplegenes.RomaexhibitresistantgenesagainstI1andPh3while advance lines (1008) had resistance against Bwr-12 and Ty-2 genes. A cross was made between these two genotypes followed by series of selection and screening till F5 generation. Stable lines by pyramiding multiple disease resistant genes I1, Ph3, Bwr-12 and Ty-2 were developed. Two lines L2P2 and L2P4 with all resistant genes were developed. After gene pyramiding, agro-morphological studies were conducted using six characters comprising vine length, fruit width, fruit length, fruit weight, number of branches per plant and number of fruits per plant. Analysis of variance followed by LSD analysis depicted significant variation among the lines developed and their parents. Line with superior traits and characters was selected and identified for yield and quality enhancement programs. Data was also recorded for agro-morphological traits of all the germplasm. The data included 25 qualitative and 14 quantitative characters. Cluster analysis revealed grouping of genotypes on the basis of similarities. A total of 46 qualitative traits were divided into six clusters comprising 9, 13, 12, 7, 4 and 1 genotype in cluster I to VI respectively. Similarly 14 quantitative traits formed five clusters comprising 2, 13, 21, 3 and 7 genotypes in Group I to V respectively. Principal Component Analysis (PCA) showed positive contributionofagronomicandfruittraits.PCAextractedfivePrinciple Components having eigen values greater than 1 and accounted for 69.83% of totalvariation.PCIexplained 25.87%of totalvariation,PCIIcontributed 40.08%, PC III accounted 52.96% while 61.80% and 69.83% was contributed by PC IV and PC V respectively.Complex pattern of association was revealed by correlation studies. Path analysis showed that selection of genotypes with multiple traits and greater equatorial fruit diameter exerts positive effects in meeting the target of yield improvement. More than 50% of enough variation was investigated in the selected germplasm, which can be employed for different vital combinations as well in future breeding programs.