Bartonella is a species-rich bacterial genus that infects a wide variety of wild and domestic animals, including rodents. Despite high levels of murid rodent diversity in Africa, associated Bartonella prevalence and diversity remains understudied, particularly within the southern African subregion. To address this, we sampled endemic four-striped mice, Rhabdomys pumilio, from three rural and two urban localities in the Western Cape Province, South Africa. PCR screening and multilocus sequence analysis inclusive of five genome regions (gltA, nuoG, ribC, rpoB, and ITS), were respectively used to evaluate Bartonella status and diversity in these synanthropic rodent populations. An overall infection rate of 15% was recovered, ranging from 0% for an urban locality to 36.4% for a rural locality, consistent with the higher flea abundance recorded at the latter sites. Nucleotide sequencing and phylogenetic analyses confirmed the presence of three distinct Bartonella lineages (I–III), with lineages II and III grouping with bartonellae previously detected in R. pumilio from nature reserves in the Free State Province of South Africa, and lineage I being novel and sister to Bartonella strains identified previously in Micaelamys namaquensis. Our results indicate significant landscape effects on infection rates, highlight differential PCR assay performance, and identify three host-associated Bartonella lineages in Rhabdomys from South Africa.