Blood samples collected in four Amerindian French Guiana populations (Palikur, Emerillon, Wayampi and Kali'na) in the early 1980s were screened for selected mtDNA and Y-chromosome length polymorphisms, and sequenced for the mtDNA hypervariable segment I (HVS-I). In addition, two other Amerindian populations (Apalaí and Matsiguenga) were examined for the same markers to establish the genetic relationships in the area. Strong dissimilarities were observed in the distribution of the founding Amerindian haplogroups, and significant p-values were obtained from FST genetic distances. Interpopulation similarities occurred mainly due to geography. The Palikur did not show obvious genetic similarity to the Matsiguenga, who speak the same language and live in a region from where they could have migrated to French Guiana. The African-origin admixture observed in the Kali'na probably derives from historical contacts they had with the Bushinengue (Noir Marron), a group of escaped slaves who now lead independent lives in a nearby region. This analysis has identified significant clues about the Amerindian peopling of the North-East Amazonian region.