The aim of this study is to explore human leukocyte antigen (HLA)-DQ variability in two populations (Cayapas Amerindians and Afro-Ecuadorians) who live near one another along the Cayapa River and who are exposed to the same environmental stresses, such as infection by Onchocerca volvulus. HLA-DQA1 and HLA-DQB1 of 149 unrelated individuals (74 Cayapas and 75 Afro-Ecuadorians) have been analyzed. HLA high-resolution molecular typing was performed by sequence-based typing, sequence-specific oligonucleotides hybridization and sequence-specific primer (SSP) amplification. The comparison between affected (cases) and unaffected people (controls) in both populations shows the key role of several HLA-DQA1 alleles in susceptibility and protection against onchocerciasis. In both populations, there is strong evidence related to the protective role of DQA1*0401 against onchocerciasis. Alleles HLA-DQA1*0102 and *0103 seem to represent risk factors in Afro-Ecuadorians, while HLA-DQA1*0301 is only a suggestive susceptibility allele in Cayapas. These findings represent new positive/negative associations with onchocerciasis in South America, whereas previous findings pertained only to African populations.