Phylogenetic studies of the HIV-1 gene sequences isolated from Korean patients have suggested that most of Korean isolates belong to the subtype B strain. This study aims to characterize the Korean clade by molecular phylogenetic analysis using all of the Korean nef gene sequences registered in the NCBI GenBank (N=422), in addition to 41 reference strains and 94 foreign isolates. Through phylogenetic analyses, we verified that most of the Korean isolates belonged to the subtype B, where 78.8% are clustered exclusively of foreign isolates. This cluster has been named the Korean clade subtype B (KcB) in order to distinguish it from other subtype B clusters. Genetic distance analysis suggested that the KcB cluster was more homogeneous and clearly distinctive from the non-Korean clade subtype B (NKcB). Comparison of consensus amino acid sequences from KcB and NKcB revealed that characteristic KcB signature amino acid patterns composed of 11 amino acid residues, whose frequencies in the KcB were significantly higher than in the NKcB. The KcB signature amino acid residues were critical in identifying KcB from NKcB, since substitution of the NKcB sequences with KcB signature amino acids relocated them to the Koran clade, and vice versa.