Rabies is an important public and veterinary health threat in South Africa. The genus Lyssavirus is composed of 12 species including classical rabies virus (RABV, genotype 1), Lagos bat virus (LBV, genotype 2), Mokola virus (MOKV, genotype 3), Duvenhage virus (DUVV, genotype 4), European bat lyssavirus type-1 and type-2 [EBLV-1 (genotype 5) and EBLV-2 (genotype 6), respectively] and Australian bat lyssavirus (ABLV, genotype 7). In addition, several lyssaviruses have been also recovered from Chiroptera including Aravan virus (ARAV), Khujand virus (KHUV), Irkut virus (IRKV), West Caucasian bat virus (WCBV) as well as Shimoni bat virus (SHIV). The unusual lyssaviruses (Lagos bat virus, Mokola virus and Duvenhage virus) have been identified exclusively on the African continent. The canid and mongoose rabies biotypes in genotype 1 are commonly diagnosed in the Canidae and Herpestidae species, respectively. Dog rabies is responsible for at least 90% of the human death tolls rather than by mongoose rabies biotype through case surveillance data in South Africa and this has led to the notion that canid rabies biotype is more virulent rather than mongoose rabies biotype. Therefore, this study was proposed to the difference in virulence of two rabies biotypes prevalent in South Africa. The rabies viral genome encodes five structural proteins, namely the nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G) and RNA-dependent RNA polymerase (L). Amongst the proteins, the G-protein has been found to control entry, egress and pathogenicity of RABV, and is a critical factor for death of infected cells. Several amino acid residues which drive the pathogenicity of the RABV, known as pathogenic determinants, are found on the RABV G-protein. Furthermore, the interactions between PDZ domains and PDZ-binding site (PDZ-BS), located at the carboxyl terminus of the G-protein cytoplasmic domain (Cyto-G) could be pivotal role in the determination of phenotypes depending on cellular partners recruited by the PDZ-BS of its envelope G-protein. The present study was undertaken to provide an insight into the pathogenicity of the South African RABV biotypes, namely canid and mongoose rabies biotypes. Moreover, mongoose rabies biotype recovered in a domestic dog (referred as spill over) was also included in this study. These viruses were selected to represent the two rabies biotypes based on their reactivity patterns to a panel of monoclonal antibodies (mAbs) and phylogenetic analysis using the cytoplasmic domain of the G-protein and the variable G-L intergenic region. In order to elucidate the pathogenicity of the selected RABV isolates, the nucleotide and amino acid homologies of the complete G-protein encoding gene and pathogenic determinants on the G-protein ectodomain were evaluated. Then, the chimeric G-protein constructs were generated by grafting the carboxyl terminal of the cytoplasmic domain in a virulent RABV CVS backbone (GenBank Acc. No: AF406694). These chimeric constructs were expressed in a recombinant lentivirus system and in vitro neuronal survival or death phenotypes were evaluated to determine the pathogenicity of South African RABV constructs.