Recent data shows that range expansion of the greater mouse-eared bat Myotis myotis (Borkhausen, 1797) to Central Europe occurred mainly from the Iberian glacial refugium and in a lesser extent from South-eastern Europe. Here we present sequences of the mitochondrial control region obtained from 16 localities in the Czech Republic, Slovakia, and NW Romania. From the 97 sequences, 87 were identical with the haplotype H1, the most frequent one of haplogroup A occurring throughout Western Europe, and nine sequences (eight haplotypes) differed from H1 only by one substitution. This confirms decrease of genetic variability from south to north and colonisation of Central Europe from the Iberian Peninsula. However, we found a new haplotype, which is closely related to sequences from haplogroup D so far described in the nominative form of this species only from Greece and Bulgaria, which suggests two possible scenarios. First, colonization route from the Balkan refugium existed in this species as well, which is supported also by recently published analyses of historical DNA. Second, the Balkan haplotype entered Central Europe via interspecific hybridisation with M. blythii, a species, in which the haplogroup D is the most frequent in Europe and which is known to have colonised Europe from south-east.