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The occurrence of aflatoxin M₁ (AFM₁) in samples of raw milk of buffaloes and cows from different localities in the central areas of Punjab, the province of Pakistan, was determined by using high-performance liquid chromatography (HPLC) with prior clean-up step applying immunoaffinity columns. The present study has been designed to find out the variation of levels of aflatoxin M₁ in raw milk of different localities. Feed has major role for prevalence of aflatoxin M₁ in milk and different feed regimen is being used in different areas. Total 480 milk samples were analyzed, among these 360 were of buffalo milk and 120 were of cow milk. The percentage of AFM₁ contamination in buffalo milk and cow milk was 42.5% and 52.5%, respectively. The mean value of AFM₁ was 0.027μgL⁻¹ in buffaloes’ milk and was 0.044μgL⁻¹ in cows’ milk. In both types of milk, level of AFM₁ concentration was higher in milk samples …