ABSTRACT Aspergillus flavus, the primary causal agent of aflatoxin contamination, includes many genetically diverse vegetative compatibility groups (VCGs). Competitive ability during infection of living maize kernels was quantified for isolates from 38 VCGs. Kernels were inoculated with both a common VCG, CG136, and another VCG; after 7 days (31 degrees C), conidia were washed from kernels, and aflatoxins and DNA were extracted from kernels and conidia separately. CG136-specific single-nucleotide polymorphisms were quantified by pyrosequencing; VCGs co-inoculated with CG136 produced 46 to 85 and 51 to 84% of A. flavus DNA from kernels and conidia, respectively. Co-inoculation with atoxigenic isolates reduced aflatoxin up to 90% and, in some cases, more than predicted by competitive exclusion alone. Conidia contained up to 42 ppm aflatoxin B(1), indicating airborne conidia as potentially important sources of environmental exposure. Aflatoxin-producing potential and sporulation were negatively correlated. For some VCGs, sporulation during co-infection was greater than that predicted by kernel infection, suggesting that some VCGs increase dispersal while sacrificing competitive ability during host tissue colonization. The results indicate both life strategy and adaptive differences among A. flavus isolates and provide a basis for selection of biocontrol strains with improved competitive ability, sporulation, and aflatoxin reduction on target hosts.