We used both highly variable mitochondrial and nuclear loci to investigate the large mouse-eared bat species complex in the Western Palaearctic to clarify their systematic position. Although mitochondrial lineages show no species segregation and some haplotypes are shared between Myotis myotis and Myotis blythii sensu lato, Bayesian clustering methods based on multilocus genotypes indicate highly concordant nuclear and morphological species assignment. These multilocus, nuclear analyses detected only a single putative F-1 hybrid in the extensive areas of sympatry sampled, thus confirming the biological species status of M.myotis and M.blythii s.l. We propose that the strong cytonuclear discordance in these species complex results from a combination of prior spatial isolation of the two species in different glacial refugia, followed by a succession of mitochondrial introgression events that occurred during the eastward and westward expansions of M.myotis and of M.blythii, respectively. The nuclear markers further indicate the presence of a notable genetic discontinuity within M.myotis that broadly separates populations into an eastern and a western component with an overlap zone in the Balkans. This eastern and western discontinuity is also apparent in the mitochondrial lineages with the D haplogroup largely confined to samples found in Thrace and Asia Minor. None of these genetic discontinuities correspond to the distribution of the two commonly recognized M.myotis subspecies (myotis and macrocephalicus). We also show that distinct morphological subspecies within M.blythii (oxygnathus, omari, risorius and lesviacus) in Europe and the near-East do not correlate with significant evolutionarily units, whether identified by mitochondrial or nuclear data and thus only represent local morphological variants with little taxonomic relevance.