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Buffalo flies (Haematobia irritans exigua) are ectoparasites of major animal health and production concern in north Australian beef herds. Skin lesions associated with buffalo fly infestation, cause hide damage and welfare issues and are manifested as dermatitis or ulcerated areas found most commonly near the medial canthus of the eye, along the lateral and ventral neck and on the abdomen. Buffalo flies can transmit a nematode, Stephanofilaria sp., which has been considered the main aetiological agent for buffalo fly lesions, but the role of nematodes in the development of the lesions has not been defined. To investigate the geographical distribution of Stephanofilaria , swabs were taken from the surface of lesions and buffalo flies were netted from the backs of beef cattle from 20 properties located in northern, central and southern Queensland. Swabs and buffalo flies were then tested for the presence of Stephanofilaria by qPCR. In northern and central Queensland, all properties except one, tested positive for the presence of Stephanofilaria in either buffalo flies or swabs, or in both. The infection rate varied amongst sites ranging from 0% to 100% in lesions and 0–34% in female buffalo flies. No nematodes were found in male buffalo flies. In contrast, none of the 66 lesion swabs or 1220 buffalo flies collected from southern Queensland tested positive for Stephanofilaria infection despite the frequent occurrence of lesions in the herds from which samples were collected. These findings suggest that infection with Stephanofilaria , although frequently detected, is not essential for the development of buffalo fly lesions and other factors may contribute to the initiation of lesions. This study also confirmed the potential for using surface swabs as a quicker and less invasive means of sampling lesions than dermal biopsies when testing for the presence of Stephanofilaria by qPCR, but further studies will be required to estimate the sensitivity of this technique. Understanding the pathogenesis of buffalo fly lesions will aid the development of optimal treatment and control strategies. • Haematobia flies vector Stephanofilaria nematodes found in cattle skin lesions. • Lesion swabbing and qPCR detected Stephanofilaria in lesions and flies. • In some areas Stephanofilaria was not found despite frequent occurrence of lesions. • Stephanofilaria is not always the cause of Haematobia -associated skin lesions. [ABSTRACT FROM AUTHOR]