A total of 91 Ayoreo individuals previously studied for blood groups and protein markers, living in two Bolivian and one Paraguayan communities, were extensively investigated in relation to the hypervariable 1 segment of the control and eight coding regions of their mitochondrial DNA (mtDNA). They show an extremely reduced mtDNA variability, the observed haplotypes being classifiable in just two haplogroups (C and D). They were also variously studied in relation to six Alu insertions; in this case, however, the prevalences found did not depart markedly from those obtained in other populations of this ethnic group. To assess the Ayoreo position in relation to these populations, 11 other groups that had also been studied for these systems and for blood groups and proteins were selected. The dendrograms obtained with two of the three sets of markers showed distinct patterns, but the Ayoreo were placed in a central position in both the blood group + proteins and Alu insertions trees. Therefore, they are clearly distinct in relation to their mtDNA only, suggesting a strong founder effect and/or random loss of variability in this system.