White-tailed deer, Odocoileus virginianus, is the most important game species in Venezuela. Some populations are currently threatened by overhunting and habitat loss, making it necessary for implementation of conservation programs. In this study, we employed molecular phylogenetics and population genetics principles to identify conservation units of Venezuelan populations of this species and to provide recommendations for its management. We analyzed DNA sequences—730 base pairs of the mitochondrial control region—in 26 individuals sampled from the 3 subspecies present in Venezuela. Results revealed moderate levels of genetic polymorphism. In addition, evidence of significant population structure was found. Phylogeographic analyses showed 4 lineages with the nominal subspecies O. v. gymnotis appearing to be polyphyletic. A remarkable divergence among haplotypes from Venezuela and North America was revealed in phylogenetic analyses, the former comprising a monophyletic group. The observed divergence among haplotypes from Venezuelan and North American populations was, in most cases, higher than that observed among the latter and haplotypes of O. hemionus (black-tailed deer). This result suggests that Venezuelan white-tailed deer could be considered an evolutionarily significant unit. We interpreted the results obtained within the context of climatic changes since Late Pleistocene. In addition to ecological and morphological evidence, our data suggest that O. v. margaritae and O. v. goudotii populations, the 2 subspecies considered endangered, are clearly differentiated and should be recognized as geminate evolutionary units and protected as distinct groups, even though there is no clear support for elevating these subspecies to species rank, as proposed recently.