Sixteen microsatellite markers were developed for use on two species of long-eared bats (Nyctophilus). 454 pyrosequencing of genomic DNA was conducted on N. gouldi which is listed as endangered in South Australia. Fifteen loci successfully amplified on N. gouldi while nine cross-amplified for use on N. geoffroyi. Two populations from south-eastern Australia were genotyped for each species, comprising 91 individuals for N. gouldi and 70 individuals for N. geoffroyi. There was no evidence of linkage disequilibrium and all loci displayed Hardy-Weinberg equilibrium except Nyg19 and Nyg39 which displayed evidence of null alleles in both N. geoffroyi populations. These markers will prove valuable in assessing connectivity between endangered populations of N. gouldi, and facilitate a comparative investigation into the impacts of habitat fragmentation on two vespertilionids.