Population differentiation during the ice ages, followed by range expansions has significantly contributed to the geographic distribution patterns of the genetic diversity in Europe. In this regard, the Iberian, Italian, Balkan peninsulas and Anatolia comprise important glacial refugia. In different parts of Anatolia, suture zones, where lineages that diverged in the different glacial refugia met again, were observed for several species. In this study, we investigated the mitochondrial genetic differentiation of the greater horseshoe bat, Rhinolophus ferrumequinum in southeastern Europe and Anatolia. The mitochondrial DNA analyses indicated a suture zone in central Anatolia, similar to those recorded in other animal species, showing the presence of more than one refugium within the region. The time of the split of these lineages that diverged in allopatry was dated to the Pleistocene. However, the location of this suture zone did not coincide with the Sea of Marmara, not supporting a recent hypothesis, based on microsatellite data, which states that this water body might be an impediment to post glacial gene flow in this species.