The present study was aimed to determine the presence and levels of aflatoxin M(1) (AFM(1)) in 120 natural yoghurt samples consisting of 80 samples of commercial and 40 samples of traditional yoghurt in Guilan province in the north of Iran. The occurrence and concentration range of AFM(1) in samples were determined by immunoaffinity column extraction and high-performance liquid chromatography. Analysis of yoghurts showed that all the samples were contaminated with AFM(1) in concentration levels ranging from 4.2 to 78.9 ng/kg. In general, 16 samples (13.33%) had higher AFM(1) level than the maximum tolerance limit (50 ng/kg) accepted by European Union, but the contamination level was lower than 500 ng/kg in all the samples, which is accepted by Codex Alimentarius and National Standard. The concentration of AFM(1) in 26 samples (21.66) was lower than 10 ng/kg. The range of contamination of AFM(1) was comparatively higher in traditional yoghurt (average concentration of 32.9 ng/kg) than that in commercial yoghurt (average concentration of 21.6 ng/kg; p < 0.01). Because yoghurt is the most popular dairy product consumed in Iran, the AFM(1) contamination is a serious problem for public health. This study reports the data of a first survey on the presence of AFM1 in yoghurt in Guilan, Iran.