To investigate both aflatoxin contamination in feed produced in Japan and the distribution and movement of aflatoxin-producing fungi in the feed, a survey experiment was performed. Aspergillus fungi were isolated from imported Sudan grass hay, a material used to make total mixed ration (TMR) in Japan. HPLC analysis showed that one isolate produced aflatoxins B₁ and B₂. Isolates were identified as A. flavus and A. terreus from their morphologies, rDNA-ITS sequences and aflatoxin production. No aflatoxins were detected in the Sudan grass hay source of the isolates or the fermented TMR made from the Sudan grass as one of the raw materials. The fermentation quality of the TMR was good after ensilage for over 60 days, as indicated by increased lactic acid and decreased pH. Only lactic acid bacteria and Bacillus survived in the fermented TMR; no fungi or yeasts were detected. Fermentation is important in preventing aflatoxin contamination of silage.