Nietoperzowa Cave in southern Poland has more than 30 subfossils of mouse-eared bats of known age (820 ± 25 years BP). IfDNA has been preserved in a useable fashion in these fossils, they will provide unique opportunities for studying historic population genetics of these animals. We sequenced the entire cytochrome b gene (1,140 bp) from seven subfossil and 56 contemporary individuals of mouse-eared bats from Europe and the Caucasus Mts. Our phylogenetic estimates, combined with a low level of genetic differentiation (2.7%) suggest that M. myotis and M. oxygnathus recently diverged and are distinct at the subspecies level. We also included a fragment of mitochondrial hypervariable region (292 bp) from contemporary mouse-eared bats in our analyses, and noted that among eight haplogroups recorded in Europe and the Caucasian Mts., haplogroup D (recognized as oxygnathus) probably arose in the Crimean refugium and evolved in a steppe landscape. The Balkan stock (haplogroup F) was also successful and dispersed over extended areas. Individuals possessing this haplogroup can be found from the northern part of Apennine Peninsula to southern Poland. On the other hand, during the last ice age, individuals with haplogroup A (described as myotis) most likely found refugia in Iberia. As the glaciers retreated north, these individuals migrated north of the Alps to central Europe (and then to the Balkans). As this group has much stronger affinities with forests than mouse-eared bats from southern parts of Europe, the dispersal of these individuals would have followed the northern migration of deciduous trees in this area. The Carpathian Basin is an area of mixing for several haplogroups from different refugia, including those in Iberia, Apennine Peninsula, Balkans, and the Crimea. Nuclear RAG2 sequence data revealed reciprocal hybridization events of both historic and recent origins. Our results document for the first time that both taxa were present north of the Carpathian Mts. for at least the past 800 years (ca. 400 generations). These are the first subfossil bats from which DNA has been extracted and sequenced, opening new possibilities for future research. Finally, these data highlight the importance of large phylogeographic surveys even among very common taxa.