Geographic isolation of small populations in refugia during late Pleistocene glaciations resulted in population differentiation that in some cases lead to speciation. We report the karyotype of Microtus bavaricus, an evolutionary young and threatened rodent endemic to the Alps. Our results show that the karyotype of M. bavaricus is almost identical to that of M. liechtensteini (2N = 46, NF = 54). A close relationship between the two species was also supported by phylogenetic analysis of complete mitochondrial DNA sequences for the cytochrome b gene. The cytochrome b divergence between Microtus bavaricus and M. liechtensteini was 1.7 %, the lowest estimate observed among the 14 currently recognised species of Eurasian pine voles (subgenus Terricola).