Aflatoxin M1 (AFM1) is a mycotoxin from Aspergillus flavus and A. parasiticus, classified as carcinogenic and hepatotoxic. The objective of the present investigation was to determine its presence in raw milk from north-central Ecuador, constituted by the provinces of Pichincha, Manabí, and Santo Domingo de los Tsáchilas. These areas represent approximately 30% of Ecuadorian milk production. By the end of the investigation, a total of 209 raw milk samples were collected, obtained both during the

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... dry (June and August) and rainy seasons (April and November) of 2019. AFM1 concentrations were measured with lateral flow immunochromatographic assays, and 100% of the samples were positive for this mycotoxin, presenting a mean value of 0.0774 μg/kg with a range of 0.023 to 0.751 μg/kg. These AFM1 levels exceeded the European Union regulatory limit of 0.05 μg/kg in 59.3% (124/209) of samples, while only 1.9% (4/209) exceeded the Ecuadorian legal limit of 0.5 μg/kg. By using non-parametric tests, significant differences were determined (p ≤ 0.05) between the provinces for months of study, climatic season (being higher in the dry season), and climatic region (greater in the coast region). On the other hand, there were no significant differences (p ≥ 0.05) between the types of producers or between production systems. Therefore, AFM1 contamination in raw milk does not present a serious public health problem in Ecuador, but a monitoring and surveillance program for this mycotoxin in milk should be developed to prevent consumer health problems.