Based on polymorphism of the 12S rRNA gene and RAPD markers, differentiation of 122 tortoise individuals belonging to the three species of genus Testudo (T. kleinmanni, T. marginata, and T. graeca), six subspecies of T. graeca (T. g. nikolskii, T. g. pallasi, T. g. armeniaca, T. g. zarudnyi, T. g. terrestris, T. g. ibera), and two subspecies of the Central Asian tortoise Agrionenemys horsfieldii (A. h. horsfieldii, A. h. kazakhstanica) was performed. For comparison, 32 known sequences of 12S rRNA gene (392 bp) from tortoises of the two genera inhabiting the territories of Europe, Asia, and Africa were used. In the populations of A. horsfieldii, a total of six haplotypes; including three newly described variants, were identified. In the examined tortoises of the genus Testudo, eleven 12S rRNA haplotypes were identified. One new haplotype was detected in T. kleinmanni. Among the eight subspecies of T. graeca, eight haplotypes were identified, with four newly described ones. The reported RAPD markers generally supported the reconstructions obtained with the use of the mitochondrial marker. Similarly to the 12S rRNA-based reconstructions, two independent clusters included representatives of the two genera, Agrionemys and Testudio. Among the latter, representatives of T. marginata and T. kleinmanni, as well as T. graeca, with high statistical support values, formed two reciprocally monophyletic groups. Compared to the mitochondrial markers, RAPDs more statisticallysignificantly discriminated the sample of T. g. terrestris and the four subspecies, T. g. ibera, T. g. armeniaca, T. g. pallasi, and T. g. nikolskii. In almost all cases except the representatives of T. g. ibera, the representatives of each of four subspecies formed individual subclusters. The geographical haplotype distribution patterns and possible evolutionary scenario of the origin and dispersal of tortoises of the two genera are discussed.