Three Spermophilus populations in western Turkey with two different karyotypic forms were analyzed biometrically (NTSYS) and genetically from their 16S rRNA sequence data. Two of these populations, from Turkish Thrace and southwest Anatolia, share the same chromosomal number of 2n = 40 but were previously considered to represent two closely related species, Spermophilus citellus and Spermophilus xanthoprymnus. Our study revealed a high degree of biometric and genetic similarity between these two populations, concluding that both belong to the same species, S. citellus. In contrast, the population from central Anatolia was identified as S. xanthoprymnus, which shows the diploid chromosomal number of 2n = 42 and distinct biometric and genetic differences. UPGMA cluster analysis established the close relationship between S. citellus (Turkish Thrace) and S. citellus (southwest Anatolia) with a distance of 0.84, and S. xanthoprymnus connected to this cluster with a distance of 0.93. The similar cluster was also produced in both maximum parsimony and neighbor-joining trees by genetic sequence data of 16S rRNA. Our findings confirm the importance of a recent land bridge connecting the European part of Turkey and western Anatolia. The close biometric and genetic associations indicate a rather recent immigration of S. citellus into western Anatolia via the land bridge between the Balkans and Anatolia, probably at least no earlier then the end of the Pleistocene, and S. xanthoptymnus originating from S. citellus apparently later invading the central Anatolia steppe towards eastern Turkey.