A male Myotis alcathoe was identified morphologically among 119 road traffic casualties sampled in 2007 in southern Moravia (Czech Republic). Two additional male M. alcathoe were found among six carcasses collected in 1999 on roads in northern Moravia. Identification of all three specimens was confirmed by sequencing of mtDNA. The first photo of a M. alcathoe mandible is presented. The species, described in 2001, has actually been known from 12 localities within the Czech Republic.