Stable flies are blood feeding ectoparasites of economic, veterinary and medical importance. There is scarcity of data on pathogens harboured by stable flies occurring in South African feedlots. Hence, in this study we documented stable flies occurring in three selected South African feedlots and carried out a PCR based detection for the presence of DNA of A. marginale, Lumpy skin disease virus (LSDV), Ehrlichia spp. and Rickettsia spp. The flies were captured with vavoua traps, identified morphologically as Stomoxys calcitrans which was also supported by amplification and sequencing of both 16S rRNA and COI genes which matched with relevant species on the NCBI database. A total of 53 fly pools were used for the detection of pathogen DNA. PCR showed the presence of DNA of Anaplasma marginale (22.64%, 12/53) and LSDV (15.08%; 8/53). Neither Rickettsia nor Ehrlichia spp. DNA were detected from all the sampled feedlots. In conclusion, results obtained from this study showed that S. calcitrans in feedlots is harbouring A. marginale and LSDV which suggests that they may be involved in their mechanical transmission to livestock.