Ancient DNA (aDNA) was extracted from the human remains of seventy-three individuals from the Tommy and Mine Canyon sites (dated to PI-II and PIII, respectively), located on the B-Square Ranch in the Middle San Juan region of New Mexico. The mitochondrial DNA (mtDNA) haplogroups of forty-eight (65.7%) of these samples were identified, and their frequency distributions were compared with those of other prehistoric and modern populations from the Greater Southwest and Mexico. The haplogroup frequency distributions for the two sites were statistically significantly different from each other, with the Mine Canyon site exhibiting an unusually high frequency of haplogroup A for a Southwestern population, indicating the possible influence of migration or other evolutionary forces. However, both sites exhibited a relatively high frequency of haplogroup B, typical of Southwestern populations, suggesting continuity in the Southwest, as has been hypothesized by others (S. Carlyle 2003; Carlyle, et al. 2000; Kemp 2006; Malhi, et al. 2003; Smith, et al. 2000). The first hypervariable region of twenty-three individuals (31.5%) was also sequenced to confirm haplogroup assignments and compared with other sequences from the region. This comparison further strengthens the argument for population continuity in the Southwest without a detectable influence from Mesoamerica.