Bolivia is a key region for understanding human settlement in the South-Central Andes due to its geographical location, its ethnic diversity and its rich archeology. In order to identify the prehistoric migratory routes involved in the settlement of this region, archaeological, genetic and linguistic information is confronted. The analysis focuses on ancient mitochondrial DNA variation obtained from archaeological sites from the valleys of Cochabamba in comparison with populations from the Bolivian highlands (Tiwanaku), southern Peru, and Amazonia. For comparison, mtDNA samples of Quechua and Aymara extant populations are included. The paleogenetic analyzes suggest the existence of relationships between the valleys of Cochabamba, Tiwanaku and the Amazon, verifying the placement of these valleys as transitional areas?? between the Lake Titicaca basin and the Tropical Lowlands. Moreover, when comparing the ancient mitochondrial haplogroup distribution with extant samples of Aymara and Quechua origin, a greater genetic similarity is observed with the latter one.