The molecular diversity and phylogenetic relationship of 22 HIV-1 group O strains were examined on the basis of the protease gene and the N-terminal region of gp41env. Analysis of the newly characterized protease sequences with 12 reference sequences revealed no specific clustering patterns, despite the distinct geographic locations of the specimens. In contrast, analysis of the newly sequenced gp41 sequences with 34 published sequences revealed two distinct clusters, each represented by one full-length sequence (MVP5180 and ANT-70). Further, four of the specimens classified as group O in the protease region clustered with group M in the gp41 region (three subtype A and one subtype G, respectively), suggesting dual and/or recombinant infections with HIV-1 groups M and O. The presence of two distinct clusters in the gp41 region indicates at least two possible subtypes within group O viruses, and this may provide useful information regarding molecular epidemiological studies of group O infections.