The aim of this study was to improve the official diagnostic test package in South Africa for detection of Theileria parva infections in cattle and Cape buffalo (Syncerus caffer) and to investigate the presence of cattle-type T. parva parasites in buffalo and cattle in South Africa. To improve diagnosis of T. parva infections, a T. parva-specific real-time polymerase chain reaction (PCR) assay based on hybridization probe technology was developed. Oligonucleotide primers and hybridization probes used in the assay were designed based on the 18S ribosomal RNA (rRNA) gene. The primers amplify T. parva and Theileria sp. (buffalo) DNA but the hybridization probes specifically detect T. parva amplicons. Because of the high sequence similarity between the T. parva and Theileria sp. (buffalo) 18S rRNA genes, amplification of Theileria sp. (buffalo) DNA could not be avoided; no other bovine blood pathogens tested were amplified by these primers. The real-time PCR assay demonstrated superior sensitivity compared to other molecular tests used in detection of T. parva infections, reliably detecting the parasite in carrier animals with a piroplasm parasitaemia as low as 8.79x10-4% with minute template DNA input. The assay requires less time to perform with a low risk of contamination because of the closed-tube system that does not require handling of amplicons for post-PCR analysis. The presence of cattle-typeT. parva parasites in buffalo and cattle was investigated using restriction fragment length polymorphism (RFLP) profiles of PCR products and sequences of the parasite genes which code for the antigenic proteins p67, p104, and the polymorphic immunodominant molecule (PIM). Cattle-type p67, p104 and PIM alleles were identified from three T. parva samples obtained from cattle from a farm near Ladysmith in the KwaZulu-Natal Province. These cattle-type alleles were identical to those previously identified from a cattle-derived T. parva stock, T. parva Muguga, a parasite stock that causes East Coast fever (ECF) in Kenya; however, ECF was not diagnosed in animals in this farm. Cattle-type alleles identical to those previously reported were not identified from T. parva buffalo samples, but variants of p67 allele 1 as well as p104 allele 1, both previously obtained from T. parva Muguga, were identified. It is not known if parasites that possess these variants can cause disease, and the risk of their adapting to cattle as in the case of ECF and January disease needs to be evaluated. Furthermore, these findings suggest that cattle-like alleles may not be exclusively associated with cattle-derived T. parva parasites. Most of the p67, p104 and PIM gene sequences obtained in this study were not identical to known sequences; furthermore, novel alleles were identified, demonstrating extensive genetic diversity in the South African T. parva parasite population in buffalo. The significance of the parasites that possess ‘novel’ alleles in the epidemiology of theileriosis in South Africa still needs to be determined. The identification of variants and novel alleles reveals that p67, p104 and PIM gene PCR-RFLP profiles are more complex than previously thought and the classification of buffalo- and cattle-derived T. parva parasites in South Africa based on p67, p104 and PIM gene profiles would not be possible. Identification of more reliable markers that can be directly associated with the theilerial disease syndromes remains a challenge.