OBJECTIVE: To describe and evaluate a simple procedure to identify HIV-1 co- or super-infections based on a heteroduplex mobility assay (HMA). METHODS: To identify heteroduplexes corresponding to divergent viral populations in a the same individual, HMA was applied to single DNA samples from each subject in a prospective cohort of commercial sex workers (CSW) in Bobo-Dioulasso, Burkina Faso. After denaturation and renaturation of env DNA amplicons, hybridized DNA was separated by electrophoresis through polyacrylamide gel. HIV-1 co-infections were suspected by slow migration of heteroduplex, at a level comparable to that of mixed reference strains. Following electrophoresis, DNA in bands representing heteroduplex was extracted and cloned in a plasmid vector; identification of phylogenetically distinct clones was confirmed by sequencing divergent clones identified through a second HMA step of a pair of two mixed clones. RESULTS: Among 147 HIV-1 infected CSW, four had an autologous HMA profile comparable to low mobility of hybridized DNA from mixed reference strains representing most frequent HIV-1 clades and circulating recombinant forms (CRF) prevalent in Burkina Faso. In two of them, two phylogenetically distinct HIV-1 populations were coexisting. The first woman presented with a CRF02-AG and CRF06-cpx co-infection, and the second with a CRF02-AG and a divergent virus co-infection not significantly related to any other known subtypes. In both women, retrospective analyses of stored samples by the same HMA procedure showed acquisition of a second virus concomitent with an increasing plasma HIV RNA. CONCLUSIONS: Autologous HMA procedure followed by acrylamide extraction of heteroduplexes allowed identifying HIV-1 co- and super-infections in a large cohort study. HIV-1 super-infection is not an uncommon phenomenon.