In order to facilitate taxonomic identification and assess reproductive isolation between Myotis myotis macrocephalicus and M. blythii s.l., at the interface between the Balkan Peninsula and Middle East, we analyzed microsatellite markers and a fragment of the mitochondrial hyper-variable control region. We also examined two external morphological traits of the sampled bats, length of upper tooth row (CM³) and forearm (FA) length, to assess the reliability of distinguishing them morphologically. Myotis blythii s.l. had higher allelic and haplotype richness than M. m. macrocephalicus, supporting the hypothesis that the former was present in Asia Minor before the arrival of M. myotis from Europe. The microsatellite markers analyses suggested that all sampled individuals were ‘pure’ forms of either species. Both species, however, shared a few mitochondrial haplotypes, which seem to document past introgression events rather than the recurrent interbreeding. Length of CM³ and FA increased with longitude in both species. A binary logistic regression model, using CM³ measurements and longitude of the sampling locality, correctly assigned 98% of genetically identified individuals. The sex-specific binary regression models, based on FA measurements and longitude were correct for 96% females and 98% males.