Different regions in the worldusing Stevia rebaudiana  Bertoni as sweetener crop. It is a perennial semi-shrub up to 40 cm in height. Stevia have versatile medicinal uses without any side effects. It could propagated by seed or by stem cutting. Although seed propagation is very common method, seed is not efficient because of poor germination, low fertility and incompatibility of the flowers. Destruction of the donor plant and decreased new plant number made stem cutting method is limited. In our study, Stevia in vitro propagated using nodal segments on Murashige and Skoog (MS) medium and various growth regulator concentrations [6-benzyladenine (BA), and kinetin (Kin)] The best and highest response of regenerants was in the medium contain (2.0 mg/l BA+ 0.5 mg/l Kin) which recorded (87.43), (9.07) and (4.61) for the percentage of explants, maximum total shoots number, the highest average of shoot length, respectively. Maximum average of root/shoot length and the highest percentage of root/shoot recorded in medium supplemented with 1.0 mg/l IAA (12.93, 6.89 and 98 %). The difference ratio among T1 & T2 in Constituents (%) of moisture, Fat, Ash, Crude fiber, St and Reb-A were a slight ratio thus both treated media are likely favorable in our work. Obviously, the obtained data about moisture, protein and total carbohydrate were the noticeable difference increased in T2 more than T1, these observations interpreted as evidence for the combination high ratio of protein and stevioside sweeteners might be due to the favorable media to achieve most growing performance, which reflect on dietary values. Acclimatization succeeded with a 95% survival rate of the ex-vitro plantlets at the hardening phase. Genetic stability of propagated plantlets compared with compared with mother plants was analyze using Inter simple sequence repeat (ISSR) markers. The present study provides report on the genetic fidelity of micropropagation for Stevia rebaudiana obtained from axillaries bud explants using ISSR analysis. The tissue cultured plantlets and the mother plant were identically with no variability detected.