Tabanidae (Diptera) is a diverse haematophagous fly family, known to transmit over 35 livestock pathogens both mechanically and biologically. Kruger National Park (KNP) (in Limpopo and Mpumalanga provinces), South Africa, has a high diversity of tabanids, representing 26 of the total indigenous species (n=213) present in South Africa. Little modern taxonomical work has been done on tabanids within South Africa despite their medical, veterinary and environmental importance. This study aimed to determine a cost-effective DNA extraction method as well as comparing the traditional alpha-taxonomic approach to species delimitation with molecular methods using two gene regions, mitochondrial cytochrome oxidase I (COI) and nuclear alanyl-tRNA-synthetase (AATS) of tabanids collected in KNP. Furthermore, the study aimed to elucidate the role of tabanids in the transmission of Besnoitia besnoiti as this pathogen has found to ciculate in impala (Aepyceros melampus) and blue wildebeest (Connochaetes taurinus). A comparative study on DNA extraction methods were conducted, of which the most effective method was selected for DNA extractions. Tabanids were captured in three locations within KNP. The flies were morphologically identified then homogenized. DNA was pooled for the B. besnoiti screening, followed by sequencing and phylogenetic analysis. In total, 856 flies were captured belonging to 16 species under five genera. The COI barcode indicated that some species are genetically uniform while others formed co-occurring haplotypes. This study found that COI alone was not adequate in distinguishing between all the species of the South African Tabanidae. It is apparent that the classification of Tabanidae should be placed under scrutiny. A larger sample size, especially with regards to the Tabanus genus, or the use of several markers will aid in clarifying their relationships. No B. besnoiti positives were detected in the screened tabanids. Furthermore, in-depth research should also be conducted in other regions of South Africa; not only on tabanid ecology and composition but their role as pathogen vectors.