The study of the HLA variability of Native American populations revealed several alleles specific to one or more of the Latin American indigenous populations. The analysis of Amerindian groups distributed all over the continent might inform about the area of origin and the dispersal of these alleles and shed light on the evolution of this remarkable polymorphism. Moreover, HLA alleles and haplotypes are excellent markers to understand the genetic relationships between populations. For these reasons, we characterized the HLA class II polymorphism in seven South American Amerindian populations and compared the results with those previously reported for other Amerindian groups. The Guarani-Kaiowá (n = 160) and Guarani-Nandeva (n = 87) were from the Brazilian state of Mato Grosso do Sul, the Guarani-M'byá (n = 93) and Kaingang (n = 235) from Paraná state, the Aché (n = 89) from eastern Paraguay, the Quechua (n = 44) from Andean Peru. From Amazonia, a heterogeneous group was analyzed (n = 45). The most frequent alleles and haplotypes are common also in other Amerindian populations. Each HLA-DRB1 allele was typically found in combination with just one DQA1-DQB1 haplotype, most likely as a result of some form of random genetic drift and reduced gene flow from non-Amerindians. The frequency distribution differed significantly among all populations, although differences were less pronounced between the Guarani subgroups. Marker alleles allowed an estimate of European and sub-Saharan African gene flow into these populations: Quechua 23%, Guarani-Nandeva 14%, Kaingang 7%, Guarani-M'byá 4%, Guarani-Kaiowá, Amazonia, and Aché 0%. Interestingly, the DRB1*1413 allele, previously found only among the Guarani-M'byá (frequency 15%), appeared in the Aché (8%). The relationship of the Aché to other Amerindian populations is unclear, and this finding reveals a link with the Guarani. On the basis of genetic distance and the HLA allele/haplotype set, we propose that the Aché are differentiated Tupi-Guarani group, most closely related to the Guarani-M'byá.