Stevia rebaudiana is a natural sweetener herb that is increasingly used in herbal medicines in the food and cosmetics industries. Molecular methods can be combined with morphological techniques to identify stevia genotypes as a starting material to produce more reliable bioproducts. This study evaluated the level of the genetic and biochemical diversity in various stevia genotypes using HPLC (high performance liquid chromatography) analysis and random amplified polymorphic DNA (RAPD) markers. Stevia genotypes collected from different locations of the world showed clear variations at the biochemical and genetic level in Polish climate conditions. The influence of the genotypes on the content of steviol glycosides, antioxidants, phenols, flavonoids, and tannins was analyzed using phytochemical assays. Genotypes from Morocco, Poland, Egypt, and Nigeria can be defined as samples of higher quality compared to other genotypes analyzed in terms of the amount of steviol glycosides. Considering the rebaudioside A/stevioside ratio as a selection criterion, genotypes from Australia, China, India, and Pakistan should be considered to be valuable in terms of suitability for obtaining new varieties. The present results of RAPD marker analysis indicated differential banding pattern and considerable polymorphism among all ten stevia genotypes. Genotypes from Morocco, Egypt, Poland, Nigeria, China, and India, as genetically different, can be selected for further stevia breeding programs.