The present study aimed to identify the molecular markers as well as the effects of genotype, explant and hormone balance on the ability of shoot regeneration in vitro tomato propagation. Six commercial cultivars (SM, UC97-3, CR, SQ, SSB and RS) and four hybrids (SM×CR, SSB×CR, RS×CR and SQ×CR) were used as different genotypes. Two explant types (shoot tip, eypocotyl) and three media composition [M1 (MS + BA 2 mg/l + Kin1 mg/l.), M2 (MS + BA 0.5mg/l + Kin0.5mg/l.), and M3 (MS + BA1 mg/l + Kin2 mg/l.)] affected shoot regeneration in all the tested genotypes. The three types of media promoted the shoot tip explant to produce shoots. Only M1 medium promoted the eypocotyl explant to produce shoots. Three criteria were measured on all produced plantlets (leaf number, plant height and number of shoots/ explant). Three genotype(SM, UC97-3, CR and RS) showed the highest number of shoots, number of leaves and plant height, respectively as a response to the culture medium M1 (2mg/l BA +1mg/l kin) So, they are good combiners for the production of hybrids. The hybrid (SM×CR) possessed high number of shoots.Ten ISSR primers were individually amplified to allow the differentiation of the materials under study. All ten primers generated 98 DNA bands, with an average of 9.8 per primer and 78 being polymorphic, The profiles generated by primer 17898 B (CA) 6GT contained the highest number of polymorphic bands (12 bands). All primers detected unique bands except HB13and HB10 primers. The two varieties (SM) and UC97-3 possessed highly shoot regeneration than the other genotypes and they had unique bands may be used as molecular markers for highly shoot regeneration. These molecular markers were 917 bp by primer 17899 A (CA) 6 AG, 365 bp, 333 bp and 291 bp by 17898 A(CA) 6AC and 515 bp and 343 bp by primer 17898 B (CA) 6GT. The evaluation of the dendrogram showed 79% similarity between SSB and UC97-3 cultivars and they are located in a separate group, and 82% similarity between CR, SQ, RS, SM cultivars. From the results obtained in this study, ISSR markers have a high efficiency to differentiate the tomato cultivars. Three primers i.e., 17899 A (CA) 6 AG, 17898 A(CA) 6AC and 17898 B (CA) 6GT consider as a specific primers in tomato genotypes for discovery of molecular markers to high ability shoot regeneration genotypes.