The aim of this study was to evaluate aflatoxin M1 (AFM1) contamination in milk samples in Mashhad in Iran. A total of 61 milk samples were collected from retail stores in June 2013. The occurrence and concentration range of AFM1 in the samples were investigated by ELISA technique. AFM1 was found in 53 (86%) of the examined milk samples by average concentration of 118.6ng/L and the contamination level ranging between 0-250ng/L. The concentration of AFM1 in 30 (49%) samples was lower than the Iranian national standard (100ng/L) but the mycotoxin level in all the samples was lower than Food and Drug Administration limit (500 ng/L), and only in 9 (14.7%) of the samples, the concentration of AFM1 was lower than the maximum tolerance limit (50ng/L) accepted by European Union and Codex Alimentarius Commission. This situation must be considered as a food safety concern.