The mitochondrial DNA (mtDNA) of 139 individuals from eight tribes which belong to four linguistic groups of the Brazilian Amazon Region was studied both by RFLP and by sequencing of the D-loop region. RFLP analysis showed that 41 haplotypes (29%) belonged to haplogroup A, 39 (28%) to haplogroup B, 38 (27%) to haplogroup C, 19 (14%) to haplogroup D, and 2 (< 2%) could not be assigned to any of the four haplogroups. Among the 92 individuals analyzed by direct sequencing of the D-loop region, we observed 43 different haplotypes defined by 48 polymorphic points, while one haplotype could not be assigned to any of the clusters previously described. Joint analysis of data obtained by RFLP and by sequencing of mtDNA demonstrated that, regardless of the method of analysis, the mtDNA haplotypes of contemporary Amerindians cluster into four groups, similar to those previously described, even though 7% of the total sample or 12% of the haplotypes have discrepancies between results obtained by RFLP and sequencing. In addition to supporting the prevalence of four major haplogroups among contemporary Amerindians, our data are compatible with multiple founder haplotypes in each haplogroup, based on: i) a high prevalence of unusual haplotypes: ii) presence of multiple polymorphic sites shared by different haplogroups; iii) relative differences in nucleotide diversity based on RFLP or sequencing within the different haplogroups.