We report data on the genetic variation of the Tepehua population based on 15 autosomal microsatellites. The Tepehua, whose language belongs to the Totonac family, are settled throughout the Sierra Madre Oriental in Mexico and constitute a group in demographic decline. The results suggest that the Tepehua population remained isolated throughout a large part of its history. Phylogenetic analyses performed with other indigenous and admixed populations of Mesoamerica allow us to address their biological history. The results suggest a genetic affinity between the Tepehua and the Huastecos due to their previous shared history, and a certain degree of differentiation from the Otomões groups and the Choles (who are of Mayan origin). A clear genetic differentiation is also apparent between native and admixed populations within the greater region of Mesoamerica. It is currently accepted that the genetic composition of the American populations fits a trihybrid model of admixture. The genetic structure based on comparison of 34 populations throughout the continent (9 indigenous and 23 admixed) using hierarchical cluster analysis with an explained variance of 61.17% suggests the existence of four large groups distinguished according to the degree of admixture between Amerindians, Europeans, and Africans.