Aflatoxin M1 (AFM1) is a hydroxylated metabolite of aflatoxin B1 (AFB1) that can be excreted in milk of cows after consuming contaminated feed. The aim of this study consisted of a field monitoring to assess the contamination levels of AFB1 in 60 feed samples from two feeding systems for high-yielding dairy cows and of AFM1 in the corresponding raw milk samples. The aflatoxins were analyzed by in-house validated methods based on high-performance liquid chromatography (HPLC) with fluorescence detection. AFB1 was detected in 55% of feed samples (mean 0.61 μg/kg, with 2 samples exceeding the European Union (EU) maximum level set at 5 μg/kg), with greater incidence and concentration in compound feed than in unifeed rations (p n = 22), the ratio AFM1/AFB1 exceeded the European Food Safety Authority (EFSA) estimated 6% threshold for high-yielding dairy cows.