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Mesoamerica is a cultural and geographic region with a northern boundary adjacent to the American Greater Southwest and a southeastern boundary that includes the Maya area, which is adjacent to the Caribbean. These regions are of interest to analyze genetic structure, ancestry, and gene flow between the ancient populations. We identified the mitochondrial DNA haplogroups and haplotypes in 19 colonial and 10 pre-Columbian Maya from Xcaret and 6 Paquimé individuals. We analyzed our data together with 603 ancient individuals and with 95 colonial and 502 pre-Columbian individuals. The results show clear genetic differences among Mesoamerica, American Greater Southwest, and Caribbean regions. High frequency of haplogroup A in Paquimé and Mine Canyon and the distribution of its haplotypes in the networks suggest these populations are probably genetically related with both Mesoamerican and the American Greater Southwest populations. The genetic structure of the Maya is due to common ancestry, and it was maintained by geographic isolation and gene flow mostly between Mayan populations. The Spanish conquest did not change this structure in the Maya from Xcaret, Quintana Roo. Although populations from Central Mexico are not genetically homogeneous, they are clearly different from Maya. Teotihuacan and Cholula were contemporary cities that allied to control the region, but they show genetic differences that could be related to a distant common ancestry; they probably descended from the same group but separated very early, before their arrival in Central Mexico.