The Aztec empire emerged in AD 1428 as a result of the triple alliance among the city-states of Tenochtitlan, Texcoco, and Tlacopan. Although it is well documented that the Aztecs conquered numerous polities in the Basin of Mexico over the next 100 years, the demographic consequences of this expansion remain unclear. At the influential Otomi city-state of Xaltocan, for example, colonial documents suggest that the Aztec conquest led to a replacement of the original Otomi population, whereas archaeological finds suggest that a significant portion of the original population may have remained at the city under Aztec rule. To help resolve questions about Xaltocan’s population history during this period, I extracted ancient DNA from 21 individuals that can be divided into two temporal subpopulations (roughly predating and postdating the hypothesized replacement event). I determined mitochondrial DNA haplogroups through RFLP analyses and constructed haplotypes based on 372 bp of HVR1 sequence. Statistical analyses show significant differences between the mitochondrial composition of the two subpopulations. Altogether, the results of this study support the hypothesis that matrilines at Xaltocan underwent a significant replacement event following the Aztec conquest, and they suggest that the Aztec expansion may have had a substantial genetic impact on certain Mesoamerican populations. / text