In contrast to the Eastern Palaearctic region a high degree of cryptic diversity was discovered among temperate bats of the Western Palaearctic region in the last ten years. Climatic oscillations caused severe changes in the distribution of species throughout the Palaearctic region during the Pleistocene. Exploring multiple taxa can help to understand general evolutionary differentiation processes. In the present study genetic variation within and among 94 Mongolian vespertilionid bats of six genera (Hypsugo, Eptesicus, Vespertilio, Myotis, Plecotus, and Nyctalus) was screened by sequencing a 798 bp fragment of the mitochondrial ND1 gene and then subsequently compared with those of Western Palaearctic taxa. This allowed first insights in the differentiation among a wide range of bats across the Palaearctic region. A total of 16 distinct mitochondrial lineages were found in Mongolia. Thirteen lineages differed by at least five percent sequence divergence from Western Palaearctic species. Only three lineages (Eptesicus nilssonii, Vespertilio murinus, and Nyctalus noctula) showed lower divergence values. Our data demonstrate a substantial differentiation between most Western and Eastern Palaearctic vespertilionid bats. Estimations of divergence times showed that most divergence appeared prior to the Pleistocene, but current distributions of bats were most likely shaped by the usage of multiple refugia during glaciations.