Tuberculosis caused by Mycobacterium bovis is recognized worldwide as a significanthealth risk in domestic cattle, farmed and wild animal species as well as in humans. Wecarried out spoligotyping and variable number of tandem repeat (VNTR) typing methodsto characterize 490 M. bovis isolates from livestock (cattle, n = 230; pig n = 1) and wildlifespecies (n = 259) originating from different farms and regions in South Africa, with the aimto further establish the genetic diversity of the isolates, study the population structureof M. bovis and elucidate the extent of interspecies transmission of bovine tuberculosis.A total of ten spoligotype patterns were identified, two of which were novel (SB2199and SB2200) and reported for the first time in the literature, while VNTR typing revealeda total of 97 VNTR profiles. Our results showed evidence of clonal expansion for someancestral strains as well as co-infections with two or three M. bovis strains on some of thecattle and game farms, which suggested independent introductions of infected animalsfrom epidemiologically unrelated sources. Five spoligotypes and nine VNTR profiles wereshared between cattle and wildlife. Our findings showed that besides cattle, at least16 different animal species in South Africa are infected with bovine tuberculosis, andhighlight a strong evidence of inter and intra-species transmission of M. bovis. Infectionof the blue wildebeest (Connochaetes taurinus) with M. bovis is described for the first timein this report. This update in epidemiological information raises concerns that bovinetuberculosis has increased its spatial distribution in South Africa and is also affecting anincreasing number of wildlife species compared to ten years ago.