Aflatoxins are toxic substances can contaminate food crops and pose a serious health threat to humans and livestock. Aflatoxinis are produced by Aspergillus flavus and Aspergillus parasiticus and these moulds can grow in improperly stored feed and feed with inferior quality of ingredients. Species identification of Aspergillus from the animal feed will give signs and types of mycotoxins produced. Therefore, ensuring adequate resources are available for testing and early diagnosis is very important for reducing serious health threat to humans and livestock. In this study, we isolated fungal isolates from five types finished feed for farmed Nile tilapia to identify fungal isolates to species level. Molecular method of assessment was done by using ITS1and ITS4 primers in the polymerase chain reaction (PCR) and sequences were analyzed by basic local alignment search tool analysis (BLAST). Our results indicate that all the five fungal isolates from Nile tilapia feed showed compatible (100%) homology with other Aspergilus flavus isolates on National Centre for Bioinformatics. Phylogenetic analysis was studied based on Neighbor-Joining and Maximum Likelihood methods with 1000 bootstrap values, using ITS region and the sequences showed that the A. flavus isolates from tilapia feed and other A. flavus isolates were clustered in the same clade.