Genetic variability induced by cell and tissue culture techniques provides a new source of diversity for improvement of ornamental plants species. For this purpose, the effect of Thidiazuron (TDZ) on callus induction and number of subcultures of Salvia splendens was studied. The results showed that the MS medium supplemented with TDZ (0.5 mg/l) and IAA (0.1 mg/l) produced the highest percentage for explants producing callus (60%). For shoot multiplication, the best results were achieved when shoot tips were cultured on MS medium supplemented with TDZ (0.2 mg/l) alone. For subculture the same concentration of TDZ (0.2 mg/l) in media of eleven subcultures were performed at 21-day intervals over a total period of 231 days. For molecular variation detection, SCoT molecular markers were employed. Five SCoT primers were used and a total of 24 bands were obtained. The results showed high level of polymorphism between parent and their regenerated plants (subculture No. 11). These markers revealed distinctly different patterns of DNA fragments in all somaclones, and some distinctive bands were linked to the regenerated plants. For rooting, the best rooting percentage (100.00%), highest number of roots/shoot (7.00) and root length (2.58 cm) were observed when the shoots were cultured on MS hormone-free medium supplemented with 15 g sucrose/l. Plantlets were successfully acclimatized on Peat moss + perlite (1:1) with a survival rate of 100 %.