Artificial selection related with important agronomic characteristics of Stevia rebaudiana may cause genetic divergence and formation of genetically structured populations with genetic uniformity or diversity within cultivars. Current study employed inter simple sequence repeats of DNA (ISSR markers) to assess genetic diversity within and among a single cultivated population maintained through sexual propagation (SR1) and four cultivated populations generated by artificial selection and maintained by vegetative propagation (SR2-SR5). Highest polymorphism rate was reported in SR1 (89.24%), whilst the lowest rate of polymorphism occurred in SR2 (60.13%). ISSR markers revealed that selection of plants with traits of vegetative-propagated interest may lead towards the generation of genetically more uniform DNA-level populations, while plants maintained by sexual propagation have high genetic variability. High estimated genetic divergence level indicated that the five areas of stevia form genetically structured populations. SR2 and SR4 are constituted by plants more homogeneous at DNA level for the selected characteristics than plants of SR3 and SR5 populations. Predominant and homogeneous genotypes selected at SR2 and SR4 areas could be valuable for tracing strategies to obtain stevia plants with the desirable agronomic characteristics through crosses between contrasting individuals in future breeding programs.