AbstractOdocoileus virginianus (white-tailed deer) and O. hemionus (mule deer) are sympatric across much of North America. Molecular evidence suggests that up to 24% of individuals in some populations are a product of hybrid ancestry. Several studies have alluded to ancient and recent introgression between Odocoileus spp.; however, no divergence dates were proposed. Herein, phylogenetic analyses of DNA sequences obtained from the mitochondrial Cytochrome b gene in 690 individuals identified three clades corresponding to black-tailed deer, white-tailed deer, or a unique combination of both white-tailed deer and mule deer. White-tailed deer and mule deer diverged from a common ancestor of approximately 3.13 mya followed by an ancient hybridization event of approximately 1.32 mya, in which the white-tailed deer mitochondrial genome was “captured” by mule deer. This hybridization event produced a novel haplogroup for white-tailed deer and mule deer located west of the Appalachian Mountains and east of the Cascade Range, south to Veracruz, Mexico, and north to the Yukon Territory, Canada. The ancestral mule deer-like mitochondrial genome appears to be restricted to black-tailed deer distributed along the western portion of the Cascade and Sierra Nevada Ranges of the United States and Canada, whereas the ancestral white-tailed deer-like mitochondrial genome is restricted to the eastern United States and portions of Latin America and Caribbean regions. The “captured mitochondrial genome” has continued on an independent evolutionary trajectory and represents a unique and broadly distributed haplogroup that is 7.25% and 2.84% different from the ancestral mule deer and ancestral white-tailed deer haplogroups, respectively.