Aspergillus flavus is distributed throughout the world, but are more common in warm climate zones, thus the risk of infection is more in hot and dry weather conditions like that of Gujarat. We investigated defects in the aflatoxin gene cluster in 81 non-aflatoxigenic A. flavus isolates collected from different peanut growing fields of Gujarat state (India). PCR assays using aflatoxin-gene-specific primers grouped these isolates into 53 deletion patterns and three groups. It was revealed that 84% (group 2) and 11% (group 3) of the non-aflatoxigenic isolates had 1-7 and 19-27 gene-defects respectively. However 5% (group 1) of the non-aflatoxigenic isolates were found to have no gene-defects. No isolate was found defective for all the genes of aflatoxin biosynthesis gene cluster and flanking region. Thus, deletions in the gene-cluster among non-aflatoxigenic A. flavus isolates are not unusual, and the deletion patterns were quite diverse. Screening of isolates using gene specific PCR was found quite effective for the identification of gene-defects in A. flavus. Four most defective, group 3 isolates were identified, which can be used as bio-control agents in the form of “cocktails” for the Indian peanut growing areas.