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Aspergillus flavus is an agriculturally important fungus that causes ear rot of maize and produces aflatoxins, of which B 1 is the most carcinogenic naturally-produced compound. In the US, the management of aflatoxins 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. This study examined up to 27,529 single-nucleotide polymorphisms (SNPs) in a total of 815 A . flavus isolates, and 353 genome-wide haplotypes sampled before biocontrol application, three months after biocontrol application, and up to three years after initial application. Here, we report that the two distinct A . flavus evolutionary lineages IB and IC differ significantly in their frequency distributions across states. We provide evidence of increased unidirectional gene flow from lineage IB into IC, inferred to be due to the applied Afla-Guard biocontrol strain. Genetic exchange and recombination of biocontrol strains with native strains was detected in as little as three months after biocontrol application and up to one and three years later. There was limited inter-lineage migration in the untreated fields. These findings suggest that biocontrol products that include strains from lineage IB offer the greatest potential for sustained reductions in aflatoxin levels over several years. This knowledge has important implications for developing new biocontrol strategies.