Objective The purpose of this study was to examine South American population structure and prehistoric population displacements prior to the Spanish conquest, utilizing mitochondrial DNA haplogroups of extant mixed populations from Mexico, Costa Rica, Venezuela, Colombia, Ecuador, Peru, Bolivia, Brazil, Argentina, and Chile. Method Relative frequencies of four pan‐American haplogroups, obtained from published databases, were analyzed to evaluate patterns of variations, population structure and possible prehistoric migration pathways. Results Patterns of mtDNA variation verify biogeographic drift processes and possible migratory pathways. Conclusions We propose an updated model of South American colonization that is fully compatible with previous studies based on autosomal, mtDNA, and Y chromosome variation and with archaeologically‐derived culture history.