The genetic variation in a Chibcha-speaking Amerindian tribe from lower Central America, the Huetar, was analyzed using nucleotide sequences of the hypervariable segments of the mitochondrial DNA (mtDNA) control region, the frequencies of 10 Amerindian-specific mtDNA haplotypes, and the regional distribution of private protein polymorphisms. The sequencing of 713 base pairs (bp) in the control regions of 27 individuals revealed 11 distinct lineages. These were defined by 24 variable sites and a 6-bp deletion between nucleotide pairs (np) 106 and 111. The 6-bp deletion is a new mtDNA marker that will be valuable for Amerindian taxonomic research. Control region sequences and mtDNA haplotype analyses reveal that Huetar mtDNAs are distributed in "Amerindian clusters" A, B, and D. A maximum-likelihood phylogenetic tree suggests a single origin for the 6-bp Huetar deletion in the sample. mtDNA haplotype analysis and the presence of previously characterized private protein variants (PEPA*F, TF*DCHI, and the absence of DI*A) show that the Huetar harbor polymorphisms of considerable antiquity, suggesting an early divergence from the regional founder gene pool for this population. The data also reflect a drastic constriction in population size, an evolutionary event with a proposed central effect on Huetar genetic structure.