Previous studies have indicated that the common European pipistrelle bat (Pipistrellus pipistrellus) comprises two cryptic species, P. pipistrellus and Pipistrellus pygmaeus, which differ in echolocation call frequency and mitochondrial DNA sequence. However, levels of divergence based on nuclear markers have not been examined, and hence the potential for male-mediated gene flow between the species cannot be discounted. Moreover, little is known about population structure and migration patterns in either species. Here, we describe the use of microsatellites to investigate nuclear DNA differentiation between, and the pattern of population genetic structure within, the two cryptic pipistrelle species. In total, 1300 individuals from 82 maternity colonies were sampled across the British Isles and Continental Europe. We show, using multivariate analyses, that colonies of the same species are generally genetically more similar to each other than to those from the other species regardless of geographical location. Our findings support the hypothesis that the species are reproductively isolated. Significant patterns of genetic isolation by distance were identified in both species, indicating that mating may occur before any long-distance autumnal migration. The presence of a sea channel does not confer higher levels of genetic differentiation among colonies over and above distance alone in either species. Differences in genetic population structure were identified between the species, with P. pipistrellus showing a wider range of levels of genetic differentiation among colonies and a stronger relationship between genetic and geographical distance than P. pygmaeus. Differences in dispersal, mating behaviour, colony size and/or postglacial colonization patterns could contribute to the differences observed. © 2007 The Linnean Society of London, Biological Journal of the Linnean Society, 2007, 90, 539–550.