Babesia bovis outbreaks were reported in cattle alleged to be immunised with the commercially available live-blood vaccine in the Swartberg region of KwaZulu-Natal, South Africa and an investigation into the nature of parasites causing the outbreaks was carried out. The H isolate was obtained from a clinically ill animal on the Haistings farm and characterised using BvVA1 and Bv80 size analysis coupled with Bv80 and 18S rRNA V4 hypervariable region sequence analysis. In total, four South African B. bovis isolates were analysed: the vaccine stock (S) at passage 11 and 23 and field isolates H and F. The S23 strain used to infect vaccine donor animals could not be detected in the H isolate and could not be responsible for the severe disease symptoms observed in the field animals. Sequence profiles of the Bv80 and 18S rRNA V4 hypervariable regions for all detectable strains were compiled and now serve as a basis for the investigation of future babesiosis outbreaks. It was determined that the Bv80 PCR is not able to detect animals at the carrier stage of infection and that non-specific primer binding to Boophilus microplus and Boophilus decoloratus tick DNA occurs. For this reason, the Bv80 PCR is not suitable for investigating the nature of B. bovis infections in ticks. The BvVA1 PCR reaction required extensive optimisation and did not detect all strains present in the isolates and was therefore not used as a basis for strain discrimination. Microaerophilous stationary phase cultures of the vaccine strain at passage 24 (S24) and the H strain were initiated as a potential source of soluble parasite antigens. Continuous cultivation was not possible despite the alteration of a number of conditions. Currently there is no culture adapted B. bovis strain in South Africa and the availability of such a strain would form the basis of studies on the development of alternative vaccines.