We analyzed the Amerindian tribes, Guambiano, Ingano, and Paez of the southwest section of Colombia by major histocompatibility complex class II typing and blood group analysis in order to establish their genetic relationship. In addition, genetic admixture with Caucasian and African ancestry were determined based on blood group typing. The Paez showed admixture with Caucasian populations (22.4%), while the Ingano and Guambiano showed some admixture with Black populations (9.2 and 4.6%, respectively). The Ingano had MHC class II haplotypes found mainly in Amerindian and Asian populations with no evidence of class II haplotypes of African origin. MHC class II haplotypes of Amerindian and Asian populations and some haplotypes frequently found in European Caucasians and Asians and haplotypes of European Caucasians were found in Guambiano and Paez tribes. We compared our results with those previously reported for four Amerindian tribes on Northern Colombia. The presence of some MHC class II haplotypes in the Guambiano, Paez, and Ingano tribes and their absence in the Chibcha speaking groups of Northern Colombia suggest that these tribes originated, together with other Amerindians, from a separate migration or by genetic drift from an ancestral population. Therefore they are genetically distant from Chibcha speaking tribes of Colombia.