Papua New Guinea is in the midst of a generalized HIV epidemic. As part of a larger behavioral survey aiming to further characterize the HIV epidemic occurring in PNG, samples were collected from 1175 participants from seven different provinces. Seventy-one (6%) of these samples were HIV-1 positive, and 35 (49%) successfully underwent a double nested RT-PCR that was designed to amplify the C2-V4 region of the HIV-1 envelope. Sequence analysis showed that 33 (94%) samples were subtype C and the remaining 2 (6%) were subtype B. Further phylogenetic analysis demonstrated that there was no province-specific clustering among the samples and that within the global pandemic, PNG subtype C isolates most closely resembled those from East Africa.