The Great Lakes impose high levels of natural fragmentation on local populations of terrestrial animals in a way rarely found within continental ecosystems. Although separated by major water barriers, woodland deer mouse (Peromyscus maniculatus gracilis) populations on the islands and on the Upper Peninsula (UP) and Lower Peninsula (LP) of Michigan have previously been shown to have a mitochondrial DNA contact zone that is incongruent with the regional landscape. We analyzed 11 microsatellite loci for 16 populations of P. m. gracilis distributed across 2 peninsulas and 6 islands in northern Michigan to address the relative importance of geographical structure and inferred postglacial colonization patterns in determining the nuclear genetic structure of this species. Results showed relatively high levels of genetic structure for this species and a significant correlation between interpopulation differentiation and separation by water but little genetic structure and no isolation-by-distance within each of the 2 peninsulas. Genetic diversity was generally high on both peninsulas but lower and correlated to island size in the Beaver Island Archipelago. These results are consistent with the genetic and demographic isolation of Lower Peninsula populations, which is a matter of concern given the dramatic decline in P. m. gracilis abundance on the Lower Peninsula in recent years.