Multiple studies have demonstrated that European colonization of the Americas led to the death of nearly all North American dog mitochondrial lineages and replacement with European ones sometime between AD 1492 and the present day. Historical records indicate that colonists imported dogs from Europe to North America, where they became objects of interest and exchange as early as the seventeenth century. However, it is not clear whether the earliest archaeological dogs recovered from colonial contexts were of European, Indigenous, or mixed descent. To clarify the ancestry of dogs from the Jamestown Colony, Virginia, we sequenced ancient mitochondrial DNA from six archaeological dogs from the period 1609–1617. Our analysis shows that the Jamestown dogs have maternal lineages most closely associated with those of ancient Indigenous dogs of North America. Furthermore, these maternal lineages cluster with dogs from Late Woodland, Hopewell, and Virginia Algonquian archaeological sites. Our recovery of Indigenous dog lineages from a European colonial site suggests a complex social history of dogs at the interface of Indigenous and European populations during the early colonial period.