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Tilia tomentosa varieties were analyzed using Random Amplified Polymorphic DNA (RAPD) primers to determine genetic diversity. In this study, six samples provided by the Botanical Garden “Anastasie Fătu” of Iasi, Romania were compared to a variety collected from an ecosystem in Giessen, Germany. A total of 91 bands were obtained from nine RAPD primers, 79 (86.8%) of which were polymorphic. Marker data was used for a principal component analysis that showed clear differentiation among Romanian and German samples. Furthermore, Romanian genotypes were clustered together in the principal component analysis. PCA analysis was followed by an Unweighted Pair Group Method with Arithmetic Mean (UPGMA) cluster analysis which revealed two major groups. Samples from the Romanian ecosystem showed high genetic similarity. Generally, samples did not separate from each other in the UPGMA analysis, excepting the German sample.