Before the arrival of the Spaniards in Nicaragua, diverse Native American groups inhabited the territory. In colonial times, Native Nicaraguan populations interacted with Europeans and slaves from Africa. To ascertain the extent of this genetic admixture and provide genetic evidence about the origin of the Nicaraguan ancestors, we analyzed the mitochondrial control region (HVSI and HVSII), 17 Y chromosome STRs, and 15 autosomal STRs in 165 Mestizo individuals from Nicaragua. To carry out interpopulation comparisons, HVSI sequences from 29 American populations were compiled from the literature. The results reveal a close relationship between Oto-manguean, Uto-Aztecan, Mayan groups from Mexico, and a Chibchan group to Nicaraguan lineages. The Native American contribution to present-day Nicaraguan Mestizos accounts for most of the maternal lineages, whereas the majority of Nicaraguan Y chromosome haplogroups can be traced back to a West Eurasian origin. Pairwise Fst distances based on Y-STRs between Nicaragua and European, African and Native American populations show that Nicaragua is much closer to Europeans than the other populations. Additionally, admixture proportions based on autosomal STRs indicate a predominantly Spanish contribution. Our study reveals that the Nicaraguan Mestizo population harbors a high proportion of European male and Native American female substrate. Finally, the amount of African ancestry is also interesting, probably because of the contribution of Spanish conquerors with North African genetic traces or that of West African slaves.