Genetic diversity present in gene pool is an important determination for breeding programs, and characterization is useful of building crop plant collections primarily based on the knowledge of the presence of valuable genes and traits. Developing successful varieties for increasing the future yield and quality of tomato depend mainly on the genetic diversity of parents used in the breeding program. Molecular characterization of 21 tomato genotypes used in in Pakistani breeding program was studied using random amplified polymorphic DNA (RAPD) markers. Total 102 bands were amplified among 21 genotypes using 20 RAPD primers. Overall 73.5% polymorphism was shown as 75 out of 102 loci were polymorphic. High degree of divergence between varieties was indicated by low level of monomorphic bands. The number of PCR products per primer varied from 2-8 with an average of 5.1 bands per primer. Primer GL J-20 and GL C-09 produced maximum number of bands whereas the primers GL A-09 produced the lowest. The polymorphism per RAPD primer ranged from 50% to 100% with an average of 73.5%. The accumulative analysis of amplified products generated by RAPD’s was enough to assess the genetic diversity among the genotypes. The information would be helpful for formulating future breeding and genome mapping programs. This study will also work as an indicator for tomato breeders to evolve varieties with genetic diverse back ground to achieve sustainability in tomato production in the country.