Bartonellosis is a vector-borne disease that is often misdiagnosed due to a broad range of clinical symptoms, compounded by a lack of awareness regarding the prevalence, diversity and public health impacts of regional strains. Despite recent PCR-based confirmation of Bartonella in 9.7% of non-malarial, acute febrile patients in South Africa, data regarding reservoirs of infection are limited. As the majority of Bartonella species described to date are associated with rodent species globally, including zoonotic species such as B. elizabethae, and as rodent biodiversity is high in southern Africa, we evaluated Bartonella in the Tete Veld rat (Aethomys ineptus), a highly adaptable murid rodent that thrives in both natural and commensal settings. These rodents are infested with a broad range of ectoparasite species, and often occur in sympatry with Micaelamys namaquensis, an indigenous rodent previously shown to host B. elizabethae. DNA extracts from heart samples of 75 A. ineptus trapped over an eight-month period, from the Roodeplaat Nature Reserve (RNR), were evaluated using a multi-locus sequence analysis (MLSA) approach. Nucleotide sequencing and phylogenetic analyses of individual (gltA, ribC, rpoB and nuoG) and concatenated gene datasets confirmed the presence of three discrete Bartonella lineages (I-III). Lineages I and II, are genetically distinct from all currently recognised Bartonella species but cluster with strains present in other indigenous rodents from South and East Africa, whereas lineage III contained B. elizabethae, a zoonotic species associated with Rattus species globally. Records confirming R. tanezumi presence in this nature reserve, which is situated in close proximity to Pretoria, the administrative capital of South Africa, suggests the likelihood of spill-over from invasive to indigenous species. These results together with the high levels of infection (86.7%) and co-infection (33.8%), indicate that A. ineptus is a natural reservoir for multiple Bartonella species in South Africa, including one with zoonotic potential.