Introduction: In the departments of the Vaupés and Guaviare, in southeastern Colombia, in a transitional area between Amazonia and the eastern plains, inhabit indigenous groups belonging to the Tukanoan (East) and Guahiban linguistic families. Although some studies have dealt with the culture and the cosmology description of these groups, little research has been done on the biological diversity and genetic relationships of such groups. Objective: To estimate the diversity, the structure, and the genetic relationships of one Guahiban and two Tukanoan groups of the Colombian Amazonian region. Methods: Samples were collected (n = 106) from unrelated individuals belonging to the Vaupés native indigenous commu­nities. The DNA was extracted and nine autosomal microsatellites were typed. Several measures of diversity, FST, pairwise FST, and population differentiation between groups were calculated. Finally, it was estimated the genetic distances of the groups studied in relation with other Amazonian, Andean and Central American indigenous people. Results: 1. The genetic diversity found stands within the range of other Amazonian populations, whereas compared to the mestizo and afro-descendant Colombian populations, such diversity showed to be lower. 2. The structure and population differentiation tests showed two clusters; one consisting of the Vaupés Tukanoan and Guaviare Tukanoan groups, and a second one formed by the Guayabero. 3. Tukanoan groups are found to be closer related to the Brazilian Amazonian po­pulations than to the Guayabero. Conclusion: The results of this study suggest that the Guayabero group from Guaviare, are genetically differentiated from those Tukanoan groups of the Vaupés and Guaviare