Background and Aim: Zearalenone (ZEA) is a mycotoxin from the fungus Fusarium. ZEA can adopt a similar configuration to 17β-estradiol and other natural estrogens. Problems in the reproductive function of humans and animals have been reported for ZEA and its metabolites. This study aimed to determine ZEA in raw milk produced in representative milk production areas in Ecuador. Materials and Methods: A total of 209 samples were obtained in April and November 2019 (rainy season) and June and August 2019 (dry season). A competitive enzyme-linked immunosorbent assay technique was used to detect ZEA concentrations. Results: ZEA was determined in 99.5% (208 of 209) of the samples; however, all samples were below the maximum limits allowed (0.03-1 mg/L) in food for direct human consumption according to the Food and Agriculture Organization and European legislations. The mean (range) concentration was 0.0015 (0-0.0102) mg/L. The results did not vary significantly (p≥0.05) by cantons, provinces, weather, climate regions, types of producers, and production systems according to Wilcoxon and Kruskal–Wallis non-parametric tests. There were significant differences only between the months under study (p≤0.05). Conclusion: ZEA in raw milk from Ecuador does not represent a threat to public health. However, it is recommended to continue analyzing ZEA due to its presence in milk. It could also be present with other mycotoxins that cause harmful synergistic and additive effects to consumers.