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. A region of the mitochondrial CO1 gene was sequenced in 321 individuals. This dataset contains the mitochondrial gene region cytochrome oxidase subunit 1 (CO1) DNA sequences generated from 321 individual bat samples.