Conservation genetics is important in the management of endangered species, helping to understand their connectivity and long-term viability, thus identifying populations of importance for conservation. The pond bat (Myotis dasycneme) is a rare species classified as ‘Near threatened’ with a wide but patchy Palearctic distribution. A total of 277 samples representing populations in Denmark, Germany, Latvia, Hungary and Russia were used in the genetic analyses; 224 samples representing Denmark, Germany and Russia were analysed at 10 microsatellite loci; 241 samples representing all areas were analysed using mitochondrial D-loop and cytochrome B sequences. A Bayesian clustering approach revealed two poorly resolved clusters, one representing the Danish and German group and the other the Russian group. However, significantly, different pairwise FST and DEST estimates were observed between the Danish and German group, and between the Danish and Russian group suggesting a recent population structure. These conflicting results might be attributed to the effect of migration or low resolution due to the number of microsatellite markers used. After concatenating the two mitochondrial sequences, analysis detected significant genetic differentiation between all populations, probably due to genetic drift combined with a founder event. The phylogenetic tree suggested a closer relationship between Russian and Northern European populations compared to the Hungarian population, implying that the latter belongs to an older ancestral population. This was supported by the observed haplotype network and higher nucleotide diversity in this population. The genetic structuring observed in the Danish/German pond bat stresses the need for a cross border management between the two countries. Further, the pronounced mtDNA structuring, together with the indicated migration between nearby populations suggest philopatric female behavior but male migration, emphasizes the importance of protecting suitable habitat mosaics to maintain a continuum of patches with dense pond bat populations across the species’ distribution range.