Abstract We genotyped at least 12 Y-STRs in DNA samples of 986 Mestizos from five states: Aguascalientes ( n = 293), Jalisco ( n = 185), Guanajuato ( n = 168), Chiapas ( n = 170), and Yucatán ( n = 170). Powerplex-Y and AmpFℓSTR ® Y-filer ® kits were employed, offering a minimum haplotype diversity of 99.69% and 99.88%, respectively. The inclusion of additional Y-STR databases to the analyses allowed obtaining a Y-STR variability landscape from Mexico. Genetic relatedness and preliminary admixture estimates confirmed a population differentiation gradient throughout the Mexican territory: the European ancestry increments to the Northwest and, correspondingly, the Amerindian ancestry increments to the Southeast. These results must be considered for human identification purposes given that most states in Mexico do not have a proper Y-STR database.