Twelve Y-chromosomal short tandem repeats (STRs) DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS385ab, DYS437, DYS438 and DYS439 were studied in the three major ethnic groups from Ecuador: Mestizos, Native Amerindians (Kichwas, Quichuas) and Afro-Ecuadorians aiming to construct a representative database for this region in Latin America. All three populations exhibit high haplotypes diversities. Analysis of molecular variance (AMOVA) reveals significant differentiation between the Mestizos, the Kichwas and the Afro-Ecuadorians. The analysis of a hunter-gatherer group of Native Amerindians from the Amazonian provinces of Ecuador, the Waoranis (Huaorani) revealed markedly reduced haplotypes variability and a large genetic distance to the major Ecuadorian populations.