Genomes of human immunodeficiency virus type 2 (HIV-2), like those of HIV-1 or other retroviruses, are highly variable. These genetic variants have been classified into seven genetic subtypes. The average genetic divergence between different subtypes is about 20% in the gag gene, which is higher than those among HIV-1 group M subtypes. The current serological tests cannot distinguish the different subtypes from one another. To understand genetic variation, evolution, and subtype distribution of HIV-2, polymerase chain reaction (PCR) technology has been widely used. The PCR products that are amplified from highly conserved regions among all subtypes can be either cloned into plasmid vectors for sequence analysis or directly sequenced without a cloning step. Phylogenetic analysis of newly obtained sequences with reference sequences can determine the subtype classification or identify new subtypes if the sequences do not belong to any known subtypes.