The Otomí, or Hñäñhü, is an indigenous ethnic group in the Central Mexican Valley that has been historically marginalized since before Spanish colonization. To investigate the extent by which historical, geographic, linguistic, and cultural influences shaped biological ancestry, I analyzed the genetic variation of 224 Otomí individuals residing in thirteen Otomí villages. Results indicate that the majority of the mitochondrial DNA (mtDNA) haplotypes belong to the four major founding lineages, A2, B2, C1, and D1, reflecting an overwhelming lack of maternal admixture with Spanish colonizers. Results also indicate that at an intra-population level, neither geography nor linguistics played a prominent role in shaping maternal biological ancestry. However, at an inter-population level, geography was found to be a more influential determinant. Comparisons of Otomí genetic variation allow us to reconstruct the ethnic history of this group, and to place it within a broader-based Mesoamerican history.