Abstract HIV diversity reflects multifactorial evolutionary forces, but monitoring subtype prevalence may provide clues to understanding the epidemic. In the Americas HIV-1 C is present at significant levels only in the southern states of Brazil. We describe in this study the presence of the HIV-1 C pol genome in 11.6% (95 CI 6-21%) of antiretroviral-naive individuals from São Paulo, the major city of South America, and 6.8% (95 CI 4-12%) from the second metropolitan area of the State of São Paulo, Brazil. Moreover, a significant growth trend of this subtype was documented among cases failing therapy in the area. Sequences were obtained by direct nested PCR from cDNA retrotranscribed from plasma RNA. Phylogenetic and amino acid signatures support an expansion from variants previously identified in southern Brazil. The evaluation of additional genomic regions (partial gag, envelope, and/or integrase) in samples with HIV-1 C at pol showed extensive recombination with clade B, observed in 47% of ARV-naive cases. The spread of HIV-1 C locally and to other areas of South America should be monitored as it may influence the dynamics of the epidemic.