Bovine anaplasmosis caused by the intraerythrocytic rickettsial pathogen Anaplasma marginale is endemic in South Africa. Anaplasma marginale subspecies centrale (A. centrale) also infects cattle, however, it causes a milder form of anaplasmosis and is used as a live vaccine against A. marginale. There has been less interest in the epidemiology of A. centrale, and, as a result, there are few reports detecting natural infections of this organism. When detected in cattle, it is often assumed that it is due to vaccination, and in most cases it is reported as co-infection with A. marginale without characterization of the strain. In this study a total of 380 blood samples from wild ruminant species and cattle collected from Biobanks, National Parks, and other regions of South Africa were used in duplex real-time PCR assays to simultaneously detect A. marginale and A. centrale. PCR results indicated high occurrence of A. centrale infections ranging from 25-100% in National Parks. Samples positive for A. centrale were further characterized using the msp1aS gene, a homolog of msp1α of A. marginale which contains repeats at the 5' end that are useful for genotyping strains. A total of 47 Msp1aS repeats were identified which corresponded to 32 A. centrale genotypes detected in cattle, buffalo and wildebeest. RepeatAnalyzer was used to examine strain diversity. Our results demonstrate a diversity of A. centrale strains from cattle and wildlife hosts from South Africa and indicate the utility of msp1aS as a genotypic marker for A. centrale strain diversity.