The present study was carried out for developing an efficient in vitro callus induction and plant regeneration system in four different tomato genotypes (Solanum lycopersicum Mill., previous name: Lycopersicon esculentum), Advantage II, Edkawy, Castle Rock and Super Strain B, using hypocotyl and cotyledon explants. The effects of two cytokinins, BA (benzyl adenine) and Kin (kinetin), on callus induction and plant regeneration frequency were investigated when added to MS medium in combination at varying concentrations. All concentrations of the two cytokinins were suitable for callus induction and plant regeneration. The frequency of callus induction and plant regeneration from both cotyledon and hypocotyl explants reached 100% for all tested genotypes. Cotyledons produced a higher average number of shoots per explants than hypocotyls for all the genotypes in the five concentrations of combined cytokinins. The average number of shoots per explant in Super Strain B was found to be the highest (42 and 60 for the hypocotyl and cotyledon explants, respectively). Supplementing MS medium with 1.0 mg L−1 kinetin and 1.0 mg L−1 benzyl adenine was found to be optimum for producing the highest number of shoots per explant from hypocotyls and cotyledons in the tomato genotypes investigated. The proposed medium showed a significant superiority over the reference media.