Pteronotus psilotis, a mormoopid bat, is an insectivorous, gregarious and strict cave-dwelling species that is found areas between the sea level and an elevation of about 1000 masl. This species is present in diverse habitats ranging from rain forest to dry deciduous forest. Nine microsatellite loci were developed for Wagner’s mustached bat, Pteronotus psilotis using the next-generation sequencing approach, and their utility for population genetics studies was assessed. All loci were polymorphic (7–15 alleles) and characterized in 30 individuals from three P. psilotis populations, with the levels of observed and expected heterozygosity ranging from 0.280 to 0.867 and 0.584 to 0.842, respectively. One locus showed significant departures from Hardy–Weinberg expectations after Bonferroni correction. Cross-amplification in 11 other bat species was tested, for which eight microsatellites were successfully amplified, and of these seven were polymorphic. The development of these new microsatellite loci will contribute to investigations of genetic population structure, genetic diversity and gene flow in P. psilotis populations, as well as in other closely related bat species.