The mitochondrial genetic differentiation of the Mediterranean horseshoe bat, Rhinolophus euryale Blasius, 1853, was investigated in southeastern Europe and Anatolia. Mitochondrial DNA tRNA-proline and control region sequences were used for the analyses. As a result of the phylogenetic analyses, two reciprocally monophyletic clades were found with very high support. The results suggested that secondary contact after allopatric differentiation in separate glacial refugia, and subsequent range expansion was the best explanation regarding the evolutionary history of this species in the region. The geographical distribution of the haplotypes also suggested that the Balkans and the Black Sea could be representing refugia from which the region was populated. There also was evidence for population expansion following a pattern of isolation by distance, with geographically more distant samples also being genetically more differentiated.