In the Western Palearctic, Myotis myotis comprises two morphologically and genetically distinct, Anatolian (eastern) and European (western), populations. The populations are parapatric with a narrow contact zone in Eastern Thrace. Anatolian M. myotis are larger than those in Europe and their size increases eastwards. Genotypes based on seven nuclear markers distinguish between eastern and western populations and indicate that gene flow between these populations is limited and asymmetric. Within a few hundred kilometres of Eastern Thrace, the Anatolian population consists almost entirely of individuals with distinctly eastern genotypes. The European population is composed of both distinctly western individuals and some admixed individuals. Mitochondrial DNA also differs between eastern and western M. myotis, with eastern and western populations of M. myotis sharing only one of 28 unique HVII haplotypes. We propose that partial reproductive isolation between M. myotis populations results from divergent selective forces. Genetically and morphologically, these populations satisfy the criteria of distinct evolutionary significant units. We suggest to expand the distributional range of the Levantine subspecies, M. m. macrocephalicus, to Anatolia and to recognize two subspecies in the species rank of Myotis myotis: M. m. myotis in Europe and M. m. macrocephalicus in Anatolia and Levant.