The five aflatoxin (AF) biosynthetic pathway genes included regulatory genes aflR and aflS and the structural genes aflD, aflM, aflP were evaluated in 81 Aspergillus flavus isolates isolated from wheat, corn, barley and sorghum. The amounts of aflatoxin B1 (B1) produced by 81 isolates varied from 0.3 to 1.2 ppb. The isolates were placed into seven groups based on their DNA banding pattern and B1 production. Only one group their isolates were able to produce B1 and six groups their isolates were unable to produce aflatoxin B1. All aflatoxinogenic isolates¹⁴ showed in group I, representing more than (17%), amplification DNA fragments that correspond to the complete set of genes. Non-aflatoxigenic isolates showed six groups, group I constituted by twenty eight isolates (34.6%) correspond to the complete set of genes. Group II constituted by sixteen isolates (19.8%) showed four DNA banding pattern clustered in three profiles: aflD, aflM, aflP and aflS was the most frequent profile (11.1%) followed by aflD, aflM, aflR and aflS (6.2%) and aflD, aflR and aflS (2.5%). Group III constituted by twelve isolates (13.5%) yielded three DNA banding pattern grouped in two characteristic profiles: aflD, aflP and aflS (7.4%) and aflD, aflM and aflR (6.1%). Group IV seven isolates (8.6%) showed two DNA banding pattern clustered in two profiles. Group V constituted by three strains (3.7%) gave one profile with one DNA fragment specific for aflD gene. Finally, a group VI constituted by two isolates (2.5%) gave no DNA bands were found. Our data show a non genetic variability in aflatoxigenic A. flavus isolates, whereas a high level of genetic variability in non-aflatoxigenic A. flavus isolates.