Aspergillus flavus is an agriculturally important fungus that causes ear rot of maize and produces aflatoxins (AFs), of which B1 is the most potent carcinogen known. In the US, the management of AFs includes the deployment of biological control agents that comprise two nonaflatoxigenic A. flavus strains, either Afla-Guard (member of lineage IB) or AF36 (lineage IC). We used genotyping-by-sequencing to examine the influence of both biocontrol agents on native populations of A. flavus in cornfields in Texas, North Carolina, Arkansas, and Indiana.