The Virginia barrier islands lie along the seaward margin of the southern Delmarva Peninsula in the mid-Atlantic region of the United States, and collectively they comprise an internationally recognized biosphere reserve. Despite this recognition, no comprehensive checklist of mammals exists for the islands or the adjacent Delmarva Peninsula mainland. Herein we report on the species composition of terrestrial mammals of this region based on (1) a search of museum collections, (2) a compilation of literature sources and personal communications, and (3) our own extensive fieldwork (1975–2020). We documented 6 orders, 12 families, and 25 species of native terrestrial mammals. The islands are depauperate in comparison with the mainland, with several native species missing from the islands. We also documented 3 orders, 3 families, and 4 species of non-native mammals that are currently established on the islands. In addition, we herein present a historical overview of livestock species that were introduced to the islands, but (with 1 exception) no longer occur there. European settlement of the mainland and the islands began in the early 1600s; however, permanent human structures have been absent from most of the islands for the past 60+ years, leaving them undeveloped and in a natural state. These islands are undergoing rapid geological and ecological change under the pressure of accelerated sea-level rise associated with climate change. This checklist provides a foundation for future biogeogeographic studies of mammals in this region and serves as a baseline against which to measure future ecological and distributional changes.