The aflatoxigenic ability of strains related to Aspergillus section Flavi isolated from peanut seeds, corn and wheat grains collected from Sohag and Assiut Governorates, Egypt was assessed. A simple and rapid high performance liquid chromatographic method with fluorescence detection is optimized and employed for the determination of the aflatoxins in peanuts, corn and wheat. Sample were extracted and the analytes were separated within 7 min. Validation of the proposed method was assessed to take into account the effect of possible components. The recoveries were in the range 89–103% and RSDs were < 3.9%. Analysis of the extracts of A. flavus strains confirmed the results obtained on CAM agar plates as blue color fluorescence at 365 nm. All A. flavus strains isolated from peanut (14), corn (11) and wheat (12) produced aflatoxin(s) B1, B2 and/or G2. Among the 11 strains analyzed from corn, one strain (AUMC 9793) gave the highest concentration for the three types of toxins (B1, B2 and G2). However, aflatoxin G2 was detected from strains originated only from corn (6 strains) and wheat (9 strains), but was missing in all the 14 strains analyzed from peanut samples. Among peanut strains, AUMC 9779 gave the highest concentration for both aflatoxins detected (B1 & B2). From the 12 A. flavus strains from wheat grains, 8 produced the three aflatoxins (B1, B2 and G2) of which the most potent strains yielded high concentration of aflatoxins B1 & B2 (one strain) or the three toxins (3 strains).