Genetic variations in tissue-culture tomato plants has been examined by agro-morphological evaluation and molecular markers analysis. Four tomato varieties were used as donor parents, two types of explants (Cotyledone and hypocotyle) and two combinations of auxin and cytokinin with different concentrations were used for the first time to study the effect of genotype, type of explants and growth regulators on callus induction and plant regeneration in tomato. Significant differences among tomato varieties were observed in all tissue culture studied traits (percentage of callus induction, regeneration rate, number of regenerated plants and number roots per explant). These differences were depending on genotype, explant type, type and concentration of growth regulators. The best regeneration medium from each growth regulator combination distinct used for establishment of regenerated plants. Regenerated plants from each combination of growth regulators which exhibited wide variations compared with its donor parent were selected and subjected to somaclonal variation analysis using molecular markers and agro-morphological traits. Two molecular marker systems, ISSR (inter simple sequence repeat) and SRAP (sequence related amplified polymorphism) were used to analysis of somaclonal variation. These markers revealed polymorphism showing distinct different banding patterns in all somaclones, which were prominent in their differences from the donor parents. To confirm stability of these variation, regenerated plants (R₀) from donor parents were transferred to the greenhouse and the first generation of these somaclones (R₁) as well as their donor parents were evaluated for plant height, number of branches per plant, No. of inflorescences per plant, No. of flowers per inflorescence, No. of flowers per plant, number of fruits per plant and yield per plant. Generally, the results revealed that some somaclones exceeded their donor parents in one or more traits.