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In this study, a sample of 225 Guatemalan males, comprising 115 Mestizo-Guatemalan and 110 Mayan-Guatemalan, was typed for 17 Y-short tandem repeats (STRs) loci (DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS635, YGATA_H4.1 and DYS385a/b). The haplotype diversity (H=1) and discrimination capacity (96.86%) were calculated. Analysis of molecular variance (AMOVA) demonstrated a low but significant interpopulation differentiation when compared with the results obtained when we confront the Mestizo and Mayan populations with the European populations. Furthermore, the genetic variability and differences among the American, African, Asian, and European populations were analyzed with the software Statistica 9.1. In addition, the genetic distances were also calculated using other published data. Reynolds and Slatkińs genetic distance was visualized using the multidimensional scaling (MDS) analysis. All the analysis performed locates the Mayan population next to the Native American population, while Guatemalan-Mestizo population was found to be between these populations and the European population, similar to other Mestizo one. The implementation of the estimation of individual ancestry proportions of the whole population sample showed the presence of two well-differentiated population groups., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)