Surveillance for HIV infection among people at increased risk was conducted in five countries in South America. Seroprevalence studies were conducted in more than 36,000 people in Ecuador, Peru, Boliva, Uruguay, and Argentina, along with genetic analysis of the HIV-1 strains. In all countries, the prevalence of HIV-1 among men who have sex with men (MSM) was high (3-30%), whereas the prevalence among female commercial sex workers (FCSMs) was low (0.3-6%). By envelope heteroduplex mobility assay, subtype B predominated in MSM communities and in FCSWs in Ecuador, Bolivia, and Peru. A new genetic screening assay, the multiregion hybridization assay for subtypes B and F (MHA-bf), was developed to improve large-scale genetic screening in South America. MHA-bf can screen four regions of the genome for subtype B or subtype F, and thus can detect most recombinants. The sensitivity of MHA-bf when applied to a panel of pure subtypes and CRF12_BF was 100%, and 88% of unique recombinants were also detected as recombinant. Using MHA-bf, more than 80% of samples from Ecuador, Peru, and Bolivia were classified as pure subtype B, whereas in Uruguay and Argentina this proportion was only 30 to 40%. BF recombinants were the most prevalent form of HIV-1 in Uruguay and Argentina. Subtype B is the most common subtype in countries lacking injecting drug use (IDU) epidemics, whereas BF recombinants are more common in countries where extensive IDU epidemics have been documented, suggesting the ontogeny of recombinant strains in particular risk groups in South America.