Many organisms worldwide are responding to rapid climate change by shifting their geographic ranges. The white-footed mouse, Peromyscus leucopus, has expanded its range northward in Michigan and Wisconsin since 1980 and is now common in localities where it was previously unknown. To investigate the origin and history of the newly established populations, complete D-loop sequences were analyzed from 595 white-footed mice collected throughout the northern Great Lakes region. Mice from Wisconsin and the western Upper Peninsula (UP) of Michigan make up a well-differentiated lineage, while the eastern UP and the Lower Peninsula (LP) of Michigan form a second lineage. The two lineages diverged about 34,000 BP, well before they migrated to the Great Lakes region. The close genetic relationship between mice in the LP and those in the eastern UP is probably due primarily to accidental transport by humans and may have implications for the spread of Lyme disease in the Upper Midwest.