Aflatoxin M1 (AFM1) may occur in milk and milk products, resulting from the ingestion of aflatoxin B1 in feedstuffs by dairy cow. Ninety-six samples of commercial yoghurts (48 natural yoghurts and 48 yoghurt with pieces of strawberries) that are produced in Portugal were analyzed for the presence of AFM1 by immunoaffinity column extraction and high-performance liquid chromatography (HPLC). The limit of detection was 10 ng/kg. The recoveries of AFM1 from the samples spiked at levels of 10.0, 50.0, 100.0 and 150.0 ng/kg were 88.0%, 91.0%, 93.0% and 99.0%, respectively. AFM1 was detected in 18 (18.8%) of yogurt samples ranging from 19 to 98 ng/kg, and 78 samples (81.2%) did not reveal the presence of the toxin. Of the 48 natural yoghurts tested, only two (4.2%) were contaminated with 43 and 45 ng/kg of AFM1. Of the 48 yoghurts with pieces of strawberries tested, 16 samples (33.3%) contained levels ranging from 19 to 98 ng/kg; six samples (12.5%) were contaminated with low levels ranging from 19 to 35 ng/kg; four samples (8.3%) were contaminated with levels ranging from 36 to 50 ng/kg, two samples (4.2%) with levels of 51 and 65 ng/kg and four samples (8.3%) presented high contamination levels, from 90 to 98 ng/kg. This paper reports the data of the first survey on the presence of AFM1 in yoghurt in Portugal.