The Farming/Language Dispersal Hypothesis posits that prehistoric population expansions, precipitated by the innovation or early adoption of agriculture, played an important role in the uneven distribution of language families recorded across the world. In this case, the most widely spread language families today came to be distributed at the expense of those that have more restricted distributions. In the Americas, Uto-Aztecan is one such language family that may have been spread across Mesoamerica and the American Southwest by ancient farmers. We evaluated this hypothesis with a large-scale study of mitochondrial DNA (mtDNA) and Y-chromosomal DNA variation in indigenous populations from these regions. Partial correlation coefficients, determined with Mantel tests, show that Y-chromosome variation in indigenous populations from the American Southwest and Mesoamerica correlates significantly with linguistic distances (r = 0.33-0.384; P < 0.02), whereas mtDNA diversity correlates significantly with only geographic distance (r = 0.619; P = 0.002). The lack of correlation between mtDNA and Y-chromosome diversity is consistent with differing population histories of males and females in these regions. Although unlikely, if groups of Uto-Aztecan speakers were responsible for the northward spread of agriculture and their languages from Mesoamerica to the Southwest, this migration was possibly biased to males. However, a recent in situ population expansion within the American Southwest (2,105 years before present; 99.5% confidence interval = 1,273-3,773 YBP), one that probably followed the introduction and intensification of maize agriculture in the region, may have blurred ancient mtDNA patterns, which might otherwise have revealed a closer genetic relationship between females in the Southwest and Mesoamerica.