With considerable capacity for genetic diversification, new HIV-1 genotypes have been reported over the years. Three HIV-1 isolates previously genotyped as B using gag and env sequences were completely sequenced and reanalyzed. Several amino acid mutations were found in vif, rev, and nef genes but not in gag or env sequences. These alterations have not previously been reported in Hong Kong. The investigation of phylogenetic relatedness revealed that a region of the vif of the studied Hong Kong isolates subtype B cluster contains several subtype D signature amino acid residues. Several unique mutations on vif in these three isolates were also identified.