Contamination of certain foods and feeds with the highly toxic and carcinogenic family of Aspergillus mycotoxins, the aflatoxins, can place a severe economic burden on farmers. As one strategy to reduce aflatoxin contamination, the non-aflatoxin-producing A. flavus isolate AF36 is currently being applied to agricultural fields to competitively exclude aflatoxin-producing Aspergillus species. We now show that the polyketide synthase gene (pksA) required for aflatoxin biosynthesis in AF36, and in other members of the same vegetative compatibility group, possesses a nucleotide polymorphism near the beginning of the coding sequence. This nucleotide change introduces a premature stop codon into the coding sequence, thereby preventing enzyme production and aflatoxin accumulation.