Genetic diversity levels, effective population size estimates, and population structure of the endangered Hawaiian hoary bat (Lasiurus semotus, also known as Aeorestes semotus) were examined across the islands of Hawai‘i, Maui, O‘ahu, and Kaua‘i using eighteen nuclear microsatellite loci and one mitochondrial gene from 339 individuals collected between 1988 and 2020. The study extracted DNA for population genetic analyses from tissue samples, collected from live bats captured as part of ongoing field studies or under rehabilitation care, from bat carcasses collected by local federal and state wildlife agencies and wind energy facilities, and from dried skin specimens at the Bernice Pauahi Bishop Museum. Eighteen nuclear microsatellite loci were amplified from 298 individuals. This dataset contains the microsatellite allele fragment sizes generated from 298 individual bat samples.