calorie crop and commercially used as a non-caloric sweetener for diabetic patients. It is also used as cosmetic ingredient, pickling agent, and dentifrice. Four cultivars (Spantia, Shou2A3, China, and High Sugar) of stevia were included to optimize in vitro micropropagation. Four different combinations of hormonal treatments were investigated [6-benzylamino purine (BAP) + Kinetin (Kin) (0.25 + 0.25 mg/l); Forchloefenuron (Cppu) + Kin (0.25 + 0.25 mg/l); Cppu+ Kin (0.5+0.25 mg/l); and the control medium (hormone-free)]. Out of the different media components, the hormone-free medium produced the best performance of explants. The analysis of variance showed that the control treatment was the most significant for all traits except the number of branches per cutting. Hardening of rooted plants was performed in plastic pots with 70% survival percentage during acclimatization. Molecular characterization, of the four stevia cultivars, was conducted using 11 SCoT primers. The SCoT analysis resulted in 122 amplicons, of which, 62 amplicons (51%) were polymorphic. The range of polymorphism was between 6 % and 91 %. The range of polymorphic amplicons per primer was between one and 12 amplicons. The SCoT-16 produced the highest number of polymorphic bands (12). Meanwhile, the SCoT-24 produced the least polymorphism (6 %). The current study provides a new micropropagation system with low cost, high efficiency, and hormone-free application. Additionally, the study provides the first molecular characterization of stevia using SCoT marker system. Finally, SCoT markers associated with cultivars having high and low contents of stevioside can further be validated by marker-assisted breeding studies.