BACKGROUND: Aflatoxin M1 (AFM1) is the main monohydroxylated derivative of aflatoxin B1 (AFB1) formed in liver and excreted into milk. AFM1  creates  certain hygienic risks for human health. OBJECTIVES: The aim of this study was to determine AFM1 level in raw milk samples in Yazd province. METHODS: This investigation was a descriptive-cross sectional study. Eighty raw milk samples were collected from four cities (Yazd, Taft, Mehriz and Sadogh) in Yazd province in winter and spring seasons. The concentration of AFM1 was determined by ELISA method. The analysis of the results was performed using ANOVA and Chi-square tests. RESULTS: All samples (100%) were contaminated with AFM1, with the concentrations ranging from 3.18 to 92.24 ng/l with a mean concentration of 22.07 ng/l. AFM1 level in 13.7% of raw milk samples was higher than the maximum tolerance limit of 50 ng/l accepted by the European Union (EU). The contamination level of AFM1 in winter samples (28.21 ng/l) was higher than spring samples (15.92 ng/l). Also, the highest and lowest contamination levels were observed in milk samples collected from Sadogh (mean 42.21 ng/l) and Yazd (12.79 ng/l) cities, respectively. CONCLUSIONS: Results demonstrated AFM1 was detected with a mean concentration of 22.07 ng/l in milk samples of Yazd province. Moreover, 13.7% of samples contained AFM1 at hazardous levels for human health.