We used ancient DNA analysis of seven museum specimens of the endangered North American ivory-billed woodpecker (Campephilus principalis) and three specimens of the species from Cuba to document their degree of differentiation and their relationships to other Campephilus woodpeckers. Analysis of these mtDNA sequences reveals that the Cuban and North American ivory bills, along with the imperial woodpecker (Campephilus imperialis) of Mexico, are a monophyletic group and are roughly equidistant genetically, suggesting each lineage may be a separate species. Application of both internal and external rate calibrations indicates that the three lineages split more than one million years ago, in the Mid-Pleistocene. We thus can exclude the hypothesis that Native Americans introduced North American ivory-billed woodpeckers to Cuba. Our sequences of all three woodpeckers also provide an important DNA barcoding resource for identification of non-invasive samples or remains of these critically endangered and charismatic woodpeckers.