Five isolates of Aspergillus spp. were isolated from stored wheat cereal and peanuts (groundnuts) in the El-Monofiya governorate in Egypt. The isolates were identified using morphological and microscopic properties. The ITS (internally transcribed spacer) region was found in the location of 600 bp. Results compared with data in GenBank confirmed thatthree isolates belonged to Aspergillus flavus and two isolates belonged to Aspergillus parasiticus. Three strains of Aspergillus flavus (Af1, Af2 and Af3) and two isolates of Aspergillus parasiticus (Ap4 and Ap5) were tested to produce aflatoxin on Czapek's agar medium. To distinguish between aflatoxigenic and non-aflatoxigenic isolates, TLC (thin layer chromatography) and PCR (polymerase chain reaction) were used. The TLC technique was previously used to quantify aflatoxin (B1, B2, G1 and G2) of five strainsof Aspergillus. Three strains Af1, Af2 and Af3 gave B2 (50 μg/kg). The other strains Ap4 and Ap5 gave B1 (50 μg/kg) and G1 (75 μg/kg) for Ap4 and G2 (100 μg/kg) for Ap5. The DNA from all isolates has been extracted and amplified by PCR to encode target genes for the development of toxins (omt-A). It was observed in 3 (60%) of isolates Af1, Af2 & Af3 that the genes O-methyltransferase gene (omt-A) 300 bp. In addition, four SCoT primers and six ISSR primers were used to genetically link five Aspergillus spp. All five strains were divided into two groups for ISSR and SCoT primers. ISSR and SCOT analysis yielded similar results with TLC and aflatoxin-specific genes.