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In aquaculture, Aflatoxins are the most recorded among all mycotoxins. It seems to be particularly prone to deposition in several fish tissues representing a risk for human consumption. Aspergillus flavus is the major producer of aflatoxin that commonly contaminates food and feed all over the world. A total of 57 finished fish feed samples were collected from feed factories in Cairo, Dakahlia and Kafr El Sheikh governorates, Egypt. Twenty-nine fungal isolates belonging to four species were recovered from the collected feedstock. A. flavus was the most frequently isolated species (29.82%) compared with the other fungal species which include: A. parasiticus (12.28%), F. oxysporum (7.01%) and F. sporotrichioides (1.75%). Mycotoxin profile was determined using highperformance liquid chromatography, Aflatoxins were detected in the culture filtrate of only four isolates of A. flavus out of the seventeen examined. These four isolates were deposited under the strain codes: AUMC 13909, AUMC 13910, AUMC 13911 and AUMC 13917. A. flavus AUMC13917 strain can produce both aflatoxins B1 (1.016 µg/l) and B2 (0.314 µg/l). While, the remaining three fungal strains AUMC13909, AUMC 13910, and AUMC 13911 were able to produce only aflatoxin B1 (8.082 µg/l, 1.617 µg/l & 0.141 µg/l, respectively). Also, naturally occurring aflatoxin B1 was also detected in 4 samples of fish feed and a single sample was contaminated with aflatoxin B2. Aflatoxin B1 was the prevalent mycotoxin within the collected feed samples, which is the most toxic aflatoxin chemotype. The yield of this aflatoxin differed among the tested A. flavus strains and its concentration was …