The four aflatoxin (AF) biosynthetic pathway genes included regulatory gene aflR and the structural genes aflD, aflM, aflP were evaluated in 59 non-aflatoxigenic Aspergillus flavus isolates isolated from wheat, corn and sorghum grains. The isolates were placed into four groups based on their DNA banding pattern. group I constituted by twenty seven isolates (45.8%) correspond to the complete set of genes. group II constituted by fifteen isolates (25.5%) showed three DNA banding pattern clustered in four profiles: nor-1, ver-1 and omt-A was the most frequent profile (11.9%) followed by nor-1, omt-A and aflR (8.5%) and nor-1, ver-1 and aflR (3.4%), last profile ver-1, omt-A and aflR . group III constituted by thirteen isolates (21.9%) yielded two DNA banding pattern grouped in three characteristic profiles: nor-1 and ver-1 (13.5%), ver-1 and omt-A (6.7%), nor-1 and aflR  (1.7). group IV constituted by four isolates (6.8%) showed two DNA banding pattern clustered in two profiles. Our data show a high level of genetic variability in non-aflatoxigenic A. flavus isolates.