Tabanids are haematophagous flies feeding on livestock and wildlife. In the absence of information on the relationship of tabanid flies and protozoan parasites in South Africa and Zambia, the current study was aimed at characterizing tabanid flies collected in these two countries as well as detecting protozoan parasites they are harbouring. A total of 527 tabanid flies were collected whereby 70.2% were from South Africa and 29.8% were from Zambia. Morphological analysis revealed a total of 5 different genera collected from the sampled areas namely: Ancala, Atylotus, Haematopota, Philoliche and Tabanus. DNA extracted from South African Tabanus par and T. taeniola tested positive for the presence of Trypanosoma congolense and T. theileri whilst one member from T. par was positive for T. brucei-like parasite. DNA extracted from Zambian tabanid flies tested positive for the presence of Besnoitia besnoiti at 1.27% (2/157), Babesia bigemina 5.73% (9/157), Theileria parva-like species 30.11% (30/157) and 9.82% (14/157) for Trypanosoma evansi-like species. This study is the first to report on Babesia and Theileria parasites in tabanid flies. Further investigations are required to determine the role of tabanids in transmission of the detected protozoan parasites in livestock and wildlife in South Africa and Zambia.