Colombia is a country with great geographic heterogeneity and marked regional differences in pre-Columbian native population density and in the extent of past African and European immigration. As a result, Colombia has one of the most diverse populations in Latin America. Here we evaluated ancestry in over 1,700 individuals from 24 Colombian populations using biparental (autosomal and X-Chromosome), maternal (mtDNA), and paternal (Y-chromosome) markers. Autosomal ancestry varies markedly both within and between regions, confirming the great genetic diversity of the Colombian population. The X-chromosome, mtDNA, and Y-chromosome data indicate that there is a pattern across regions indicative of admixture involving predominantly Native American women and European and African men.