The Azorean bat (Nyctalus azoreum) is endemic to the Azores archipelago and is listed as endangered due to its reduced and fragmented distribution range. We assessed genetic diversity at eight microsatellite loci in 280 individuals from 14 locations throughout six islands. Overall, we found that the Azorean bat populations are not genetically impoverished. Indeed, the number of alleles per locus ranged from 8 to 10 and the observed heterozygosity ranged from 0.77 in Terceira to 0.83 in Faial. The highest genetic diversity and level of private alleles was observed in S. Miguel, the largest island, and the closest to the mainland. Private alleles occurred at all islands except in Graciosa. Global and pairwise F ST among islands were all statistically significant, suggesting restricted gene flow. These results, together with those of factorial correspondence analysis, Bayesian clustering method, and individual assignment tests, corroborate the conclusions of a previous mtDNA based study, providing strong support for the existence of two major subpopulations: one includes all islands of the Central Group and the other corresponds to S. Miguel. Gene flow between them is very limited, suggesting that management plans should avoid translocations between these subpopulations.