During the period of the Atlantic slave trade (15th–19th centuries), millions of people were forced to move from Africa to many American destinations, changing dramatically the human landscape of the Americas. Here, we analyze mitochondrial DNA from two different American populations with African ancestry, with hitherto unknown European and Native American components. On the basis of historical records, African-Americans from Chocó (Colombia) and the Garífunas (or “Black Carib”) of Honduras are likely to have had very different demographic histories, with a significant founder effect in the formation of the latter. Both the common features and differences are reflected in their mtDNA composition. Both show a minor component (∼16%) from Native Central/South Americans and a larger component (∼84%) from sub-Saharan Africans. The latter component is very diverse in the African-Americans from Chocó, similar to that of sub-Saharan Africans, but much less so in the Garífunas, with several mtDNA types elevated to high frequency, suggesting the action of genetic drift