Stevia  rebaudiana Bertoni is a perennial herb belongs to Compositae family used as a natural sweetener for diabetic patients, and it is native to Paraguay. Moreover, the tissue culture techniques were used to identify the best medium for callus induction and the regeneration using different concentrations of phytohormones to produce a high number of shoots and roots in each genotype. Results indicate that the Murashige and Skoog (MS) or (Linsmaier & Skoog) LS medium supplemented with 1.0 mg/l  2,4-D + 0.5 mg/l BAP + 1.0 mg/l GA3 was the favorable for all calli characters and the genotype-4 was the best one for callus induction. The green embryos character was highest in genotypes-4 with the regeneration media containing MS salts + 2.0 mg/l BAP. The high percent of multiplication and elongation shoots were obtained with genotypes-5 and 3 with a medium which containing MS salts + 0.1 mg/l BAP. Finally, the medium which contained MS salts only was the best one for differentiated roots.