BACKGROUND: Previous investigations of French Guiana Amerindians performed by this group included blood group and protein genetic markers, mitochondrial DNA and Y-chromosome investigations. Molecular autosomal data and more extensive Y-chromosome determinations were lacking. SUBJECTS AND METHODS: The genetic variability of 15 autosome (ASTRs) and 17 Y-chromosome (YSTRs) microsatellite loci was studied in four French Guiana (Emerillon, Palikur, Wayampi, Kali'na) and one Brazilian (Apalai) Amerindian populations. A sixth group, the Peruvian Matsiguenga of the Maipurean linguistic family, was included in the data analysis since they could provide information about the past migration of people from that linguistic stock into northeastern Amazonia. RESULTS: Marked ASTR and YSTR variability was found, with 96% of the YSTR haplotypes being found in one population only. There was excellent agreement between the present and previous autosomal or uniparental results. Multidimensional scaling based on F(ST) genetic distances and population structure analysis revealed heterogeneity in gene distribution, with a clear difference between the Matsiguenga and Emerillon and the other groups. In the latter, Wilcoxon sign-rank test between observed and expected heterozygosity and the mode of allele frequency distribution revealed clues of a significant past genetic bottleneck. The Wayampi stand genetically closer to the Apalai, Palikur and Kali'na when examined for the autosome but not the Y-chromosome panel of markers, suggesting preferential female gene flow. CONCLUSION: The new data provided additional important information about the biological history of people from a remote South American region, indicating how gene diversity analyses can be used to increase understanding of human microevolutionary processes.