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Aflatoxin, the secondary toxic metabolite of Aspergillus species, particularly aflatoxigenic Aspergillus flavus and parasiticus , has a detrimental effect on poultry health and production. There exists some information gap about the magnitudes of aflatoxigenic Aspergillus species and aflatoxin in poultry feeds in the study area. Thus, the study was conducted to estimate the magnitude and assess the related potential factors of aflatoxigenic Aspergillus species with evaluations of the level of aflatoxin B1 in stored poultry feed at selected farms in Dire Dawa, Ethiopia. A cross-sectional study design was carried out on 374 poultry feed samples recruited by using a stratified simple random sampling technique. A pretested structured questionnaire was used to assess the level of knowledge and prevention practices associated with aflatoxin in poultry feed. The isolation of aflatoxigenic Aspergillus species was made by Aspergillus flavus parasiticus media, and aflatoxin B1 was estimated by aflatoxin B1 enzyme-linked immune sorbent assay. Results showed that the magnitude of aflatoxigenic Aspergillus species was 72.5% (95% CI: 67.6-76.9). The odds at which the species isolated were higher ( p < 0.05) in feeds stored more than two months (AOR = 2.69), the presence of rodents in the storing room (AOR = 2.67), feeds having high moisture content (AOR = 1.5), and feed ingredient types (AOR = 4.3) compared to their counter parts. Only 34.4 and 32.8 percent of the respondents have better knowledge and apply prevention practice about fungal contamination and aflatoxin production in poultry feed, respectively. The occurrence of aflatoxigenic Aspergillus species in poultry feed was associated with the presence of rodents in the feed storing room with long storing period and high moisture contents of the feed. The knowledge and prevention practices employed by farm managers and workers about fungal contamination and aflatoxin in poultry feed are found low., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2021 Ambachew Motbaynor et al.)