Island bat species are disproportionately at risk of extinction, and Hawaiʻi’s only native terrestrial land mammal, the Hawaiian hoary bat (Lasiurus semotus) locally known as ʻōpeʻapeʻa, is no exception. To effectively manage this bat species with an archipelago-wide distribution, it is important to determine the population size on each island and connectivity between islands. We used 18 nuclear microsatellite loci and one mitochondrial gene from 339 individuals collected from 1988–2020 to evaluate genetic diversity, population structure and estimate effective population size on the Islands of Hawaiʻi, Maui, Oʻahu, and Kauaʻi. Genetic differentiation occurred between Hawaiʻi and Maui, both of which were differentiated from Oʻahu and Kauaʻi. The population on Maui presents the greatest per-island genetic diversity, consistent with their hypothesized status as the original founding population. A signature of isolation by distance was detected between islands, with contemporary migration analyses indicating limited gene flow in recent generations, and male-biased sex dispersal within Maui. Historical and long-term estimates of genetic effective population sizes were generally larger than contemporary estimates, although estimates of contemporary genetic effective population size lacked upper bounds in confidence intervals for Hawaiʻi and Kauaʻi. Contemporary genetic effective population sizes were smaller on Oʻahu and Maui. We also detected evidence of past bottlenecks on all islands with the exception of Hawaiʻi. Our study provides population-level estimates for the genetic diversity and geographic structure of ‘ōpeʻapeʻa, that could be used by agencies tasked with wildlife conservation in Hawaiʻi.