During the Pleistocene, red fox (Vulpes vulpes) populations in North America were isolated in glacial refugia and diverged into 3 major lineages: the Nearctic-Eastern subclade of eastern Canada, the Nearctic-Mountain subclade of the western mountains, and the Holarctic clade of Alaska. Following glacial retreats, these genetically distinct populations of foxes expanded into newly available habitat. Along with subsequent translocation from fur farms, these expansions have resulted in red foxes now occupying most of the continent. The origin of foxes that colonized the Great Lakes Region, however, remains unknown. Furthermore, it is unclear whether contemporary populations inhabiting this region are the result of natural range expansion or if foxes released from fur farms colonized the landscape in the 1900s. To determine the origin of red foxes in the Great Lakes Region, we collected genetic samples from 3 groups: 1) contemporary wild foxes, 2) historical wild foxes collected before fur farming, and 3) fur-farmed foxes from a contemporary fur farm. We constructed a network of mtDNA haplotypes to identify phylogeographic relationships between the 3 sample groups, and examined genetic signatures of fur-farmed foxes via the androgen receptor gene (AR) associated with tame phenotypes. Historical wild foxes demonstrated natural colonization from all 3 major North American lineages, which converged within the Great Lakes Region, and contemporary wild foxes maintained the historically high genetic diversity. Most contemporary wild foxes also matched haplotypes of fur-farmed foxes; however, AR was not useful in distinguishing fur-farm origins in samples of contemporary wild foxes. Our results show that geographically disparate populations naturally merged in the Great Lakes Region before fur-farmed foxes were introduced. Due to the historically high genetic diversity in the Great Lakes Region, any introductions from fur farms likely contributed to, but did not create, the genetic structure observed in this region.