Objective: To analyze the origin, structure,relationships, and recent admixture in Mexican Native groups basedon 15 STRs commonly used in human identification. Methods: We analyzed 39 Mexican Native population samples using STR databases based on the AmpFlSTRVRIdentifiler kit (n=3,135), including Mexican-Mestizos (admixed),European and African populations, as reference. Results: Based upon effective population size (Ne) differences, Native groups were clustered into three regions: i)Center-Southeast groups, characterized by larger Ne, migration rate (Nm), genetic diversity (He), and relative homo-geneity principally in the Yucatan Peninsula; ii) Isolated southern groups from Chiapas and Oaxaca, characterizedby lower Ne, Nm, and He (i.e. higher isolation and genetic differentiation); iii) North-Northwest groups, which aresimilar to the previous group but are characterized by generating the widest gene flow barrier in the Pre-HispanicMexican territory, and currently by elevated admixture in some northern Native groups. Despite the relative congru-ence between genetic relationships with cultural, linguistic, geographic criteria, these factors do not explain thepresent-day population structure of Native groups, excepting in those linguistically related to the Mayan that showhigher homogeneity. The Isolation by distance model was demonstrated at long distances (>1,500 km), whereas geo-graphic isolation stands as a determining factor to avoid both non-indigenous admixture and bottleneck processes. Conclusions: Different dynamics of gene flow and drift were observed among Mexican Native groups, highlight-ing the geographic barriers (mountains, canyons and jungle regions) as the main factor differentiating Pre-hispanicpopulations, and eventually helping to avoid Post-European contact admixture and population bottleneck.