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To investigate the prevalence of amphistome parasites in buffaloes slaughtered at different abattoirs of Rawalpindi and Peshawar district, Pakistan. The gastro-intestinal tracts and bile ducts were examined during the period of May to December, 2017. Out of 198 buffaloes, 39 (12.8%) were infected with multiple species of amphistomes. In present investigation, three species of amphistomes viz Paramphistomum epiclitum, Gastrothylax crumenifer and Gigantocotyle explanatum were identified. The higher infection rate was found in Kundhi breed 13.6% than Azi Khali and Nili Ravi. The males buffaloes (15.1%) and age group of 2 to 4 years (21.4%) had higher infection rate as compared to females and old animals The mixed infection of amphistomes did not show significant (P>0.05) association among breed, sex, age, contact with water bodies and grazing management of animals. The present results will contribute to our understanding of the epidemiology of amphistomiasis in Pakistan for control and prevention studies.