This study compares genetic polymorphisms at the D1S80 and HLA-DQA1 loci in three Mexican Mestizo populations from three large states (Nuevo León, Jalisco, and the Federal District). Allele frequency distributions are relatively homogenous in the three samples; only the Federal District population shows minor differences of the HLA-DQA1 allele frequencies compared with the other two. In terms of genetic composition, these Mestizo populations show evidence of admixture with predominantly Spanish-European (50-60%) and Amerindian (37-49%) contributions; the African contribution (1-3%) is minor. Together with the observation that in Nuevo León, the admixture estimates based on D1S80 and HLA-DQA1, are virtually the same as those reported earlier from blood group loci, suggests that DNA markers, such as D1S80 and HLA-DQA1 are useful for examining genetic homogeneity/heterogeneity across Mestizo populations of Mexico. The inverse relationship of the proportion of gene diversity due to population differences (Gst) to within population gene diversity (Hs) is also consistent with theoretical predictions, supporting the use of these markers for population genetics studies. Am. J. Hum. Biol. 14:257-263, 2002.