Ancient DNA recovered from 57 individuals excavated by Hiram Bingham at the rural communities of Paucarcancha, Patallacta, and Huata near the famed Inca royal estate and ritual site of Machu Picchu was analyzed by polymerase chain reaction, and the results were compared with ancient and modern DNA from various Central Andean areas to test their hypothesized indigenous highland origins. The control and coding regions of the mitochondrial DNA (mtDNA) of 35 individuals in this group were sequenced, and the haplogroups of each individual were determined. The frequency data for the haplogroups of these samples show clear proximity to those of modern Quechua and Aymara populations in the Peruvian and Bolivian highlands, and contrast with those of pre-Hispanic individuals of the north coast of Peru that we defined previously. Our study suggests a strong genetic affinity between sampled late pre-Hispanic individuals and modern Andean highlanders. A previous analysis of the Machu Picchu osteological collection suggests that the residents there were a mixed group of natives from various coastal and highland regions relocated by the Inca state for varied purposes. Overall, our study indicates that the sampled individuals from Paucarcancha and Patallacta were indigenous highlanders who provided supportive roles for nearby Machu Picchu. © 2006 Wiley-Liss, Inc.