There are two rabies virus biotypes recognized in southern Africa namely; the canid and mongoose rabies virus biotypes. The host vectors of canid rabies biotype in South Africa are domestic dogs, black-backed jackals and bat-eared foxes, whereas the mongoose rabies biotype is maintained by the yellow mongoose. The canid rabies virus was introduced into southern Africa from Angola (1940s) and spread within the subcontinent, firmly establishing itself in the domestic dog population in Zimbabwe (1950s) and South Africa (1960s). Canine rabies became established in the coastal regions of South Africa (KwaZulu Natal) in 1976 where it has been problematic ever since. Historical data demonstrate that canine rabies has spread from KwaZulu Natal into the north-eastern corner of Lesotho in 1982, spreading throughout the country and reaching the western border of Lesotho and South Africa (FS province) in the mid-1980s without penetrating into this region of South Africa. In contrast, the historical evidence suggests that mongoose rabies virus existed in southern Africa in the early 1800s. Mongoose rabies was confirmed in 1928 in South Africa and since then was consistently diagnosed in the yellow mongoose with apparent spill over into domestic animals on the central plateau of South Africa. The FS province was mainly associated with mongoose rabies; however, recent studies utilizing antigenic characterization have suggested an increase of the canid rabies biotype of RABV since the late 1990s, peaking in 2002. The aim of this investigation was to better understand the molecular epidemiology of canine rabies in the FS province by establishing genetic relationships between rabies viruses obtained from FS province and Lesotho, with the purpose of determining the origin of canine rabies into the province and the radiation of mongoose rabies biotype of RABV into dog host. The coding region of cytoplasmic domain of glycoprotein gene and G-L intergenic region of 113 rabies viruses from FS province and Lesotho was amplified and sequenced. It was found that canid rabies virus isolates from the FS province and those from Lesotho were very closely related demonstrating a mean nucleotide sequence homology of 99%. This result indicated a single overlapping epidemiological rabies cycle between the two regions. The results also confirmed that the spill over of mongoose rabies virus into dog host does not establish dog to dog transmission and therefore leads to dead end infection. Therefore parenteral vaccination of domestic dogs and cats remains an important priority in any effort to control rabies in these regions.