The Azorean bat (Nyctalus azoreum), the only endemic mammal of the Azores archipelago (Portugal), diverged recently from its mainland relative, the Leisler’s bat (N. leisleri). Although the two species are phenotypically very different, mtDNA studies detected very low genetic divergence between them, which could question the validity of the species status of N. azoreum. In order to assess the genetic variability in each species and check for present levels of gene flow between the two taxa, eight microsatellite loci were genotyped and analysed. The results indicated lower genetic diversity in the insular species. Many unshared alleles were found between the two species and no evidence of migrants, which provides strong support against any contemporary gene flow between them. The species status of the Azorean bat is discussed in the light of the cohesion species concept, and we conclude that it is an isolated species with a high conservation value.