Although scattered throughout a large geographic area, the members of the Pano linguistic group present strong ethnic, linguistic, and cultural homogeneity, a feature that causes them to be considered components of a same “Pano” tribe. Nevertheless, the genetic homogeneity between Pano villages has not yet been examined. To study the genetic structure of the Pano linguistic group, four major Native American mitochondrial DNA (mtDNA) founder haplogroups were analyzed in 77 Amerindians from six villages of four Pano tribes (Katukina, Kaxináwa, Marúbo, and Yaminawa) located in the Brazilian Amazon. The central position of these tribes in the continent makes them relevant for attempts to reconstruct population movements in South America. Except for a single individual that presented an African haplogroup L, all remaining individuals presented one of the four Native American haplogroups. Significant heterogeneity was observed across the six Pano villages. Although Amerindian populations are usually characterized by considerable interpopulational diversity, the high heterogeneity level observed is unexpected if the strong ethnic, linguistic, and cultural homogeneity of the Pano linguistic group is taken into account. The present findings indicate that the ethnic, linguistic, and cultural homogeneity does not imply genetic homogeneity. Even though the genetic heterogeneity uncovered may be a female-specific process, the most probable explanation for that is the joint action of isolation and genetic drift as major factors influencing the genetic structure of the Pano linguistic group.