Invasive species are generally problematic where they occur, especially in terms of ecology, economy and disease. Members of the genus Rattus Fischer, 1803 particularly, are known as one of the most destructive invasive species to date since they cause widespread damage on terrestrial and island ecosystems. Two Rattus species have historically been reported as invasive species in South Africa, Rattus rattus Linnaeus, 1758, which has a widespread distribution throughout the country and Rattus norvegicus Berkenhout, 1769 which is primarily distributed along the coast of South Africa. A third species, Rattus tanezumi Temminck, 1844 (which forms part of the R. rattus species complex), a south-east Asian endemic, was first reported in 2005 to also occur in South Africa (and Africa). As this species is morphologically similar to R. rattus, its identification is reliant on molecular typing approaches. In the current study, molecular, morphological and disease aspects of South African Rattus were assessed. The nature and extent of variation between the three species was investigated using cytochrome b sequences and extensive mitochondrial d-loop database for comparative purposes. D-loop data identified one, four and two haplotypes for R. tanezumi, R. rattus and R. norvegicus, respectively whereas cytochrome b data identified additional haplotypes for R. rattus and R. tanezumi. Pairwise sequence divergence was highest between R. norvegicus and R. tanezumi (12.5% for D-loop and 12.0% for cyt b). Rattus norvegicus was recovered in the central parts of South Africa for the first time and occurred sympatrically with R. tanezumi at one locality, whereas Rattus rattus and R. tanezumi occurred sympatrically at three localities. The external and qualitative cranial morphology of all three species was compared in an attempt to find differences that could be used to morphologically differentiate between these Rattus species. Whereas R. norvegicus can easily be distinguished from R. rattus and R. tanezumi, there are no discernible morphological differences to distinguish R. rattus and R. tanezumi. A taxonomic synthesis and an identification key of the three species of Rattus based on qualitative morphology, molecular and cytogenetic data using genetically-identified individuals is provided. Members of South African Rattus were also found to be carriers of the bacteria Bartonella Strong et al., 1915 and Helicobacter Goodwin et al., 1989 emend. Vandamme et al., 1991. Bartonella elizabethae (Daly et al., 1993) Brenner et al., 1993, occurring in Rattus around the world was for the first time recovered from South African Rattus. This bacterium has been associated with infective endocarditis in humans and may pose a threat to immuno-compromised individuals in rural South African communities where Rattus occurs commensally. Two Helicobacter species, H. rodentium Shen et al., 1997 and H. muridarum Lee et al., 1992, were identified neither of which have known zoonotic potential. Apart from contributing to general small mammal studies in Africa, the present study may have implications in epidemiological, agricultural, biological conservation, and invasion biology research associated with problem rodents in the southern African subregion and beyond.