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Field studies were conducted to assess the occurrence of resistance to isometamidium chloride and diminazene aceturate in trypanosomes infecting cattle in Kénédougou Province of Burkina Faso. Forty-five of the 166 villages in Kénédougou were randomly sampled and visited to assess livestock numbers, trypanosomosis risk, and tsetse challenge. The proportion of cattle infections associated with drug-resistant trypanosomes was assessed in the nine villages with the highest trypanosome infection prevalence and one village with a confirmed history of drug-resistant infections. These studies showed that resistance to both isometamidium and diminazene was widespread. However, there was considerable variation between villages in drug-resistance parameters, with the proportion of treated cattle with trypanosome infections 3 months after isometamidium prophylaxis varying from 6.9 to 63.8% and the proportion of cattle having infections 2 weeks after treatment with diminazene varying from 0 to 36.8%. The demonstration of widespread resistance to both isometamidium and diminazene has important implications, as administration of trypanocides is the most commonly employed method to control trypanosomosis in this area.