HIV-1 Vif, Vpr, and Vpu proteins have a profound effect on efficient viral replication and pathogenesis. This study describes the genotypic characterisation of vif , vpr and vpu from 20 South African HIV-1 subtype C primary isolates, and extensive analysis and comparison of known motifs. All HIV-1 subtype C Vif, Vpr and Vpu proteins revealed the presence of highly conserved structural and functional motifs similar to other sub-types, for example, the Vif-APOBEC3G interaction domains. However, several differences were noted when these sequences were compared to subtype B, such as the presence of the LRLL motif which has been implicated in targeting subtype C Vpu predominantly to the cell surface, instead of the Golgi apparatus. A better understanding of the structure/function relationship of these proteins may lead to the development of new classes of antiviral drugs. These results indicate that antiviral drugs that target the conserved functional domains within Vif, Vpr or Vpu could be active against all circulating subtypes, including HIV-1 subtype C.