Technical Abstract: Species in Aspergillus section Flavi commonly infect agricultural staples such as corn, peanuts, cottonseed, and tree nuts and produce an array of mycotoxins, the most potent of which is aflatoxin. Aspergillus flavus is the dominant aflatoxin-producing species in the majority of crops. Populations of aflatoxin-producing fungi may shift in response to: (1) clonal amplification that results from strong directional selection acting on a nontoxin- or toxin-producing trait; (2) disruptive selection that maintains a balance of extreme toxigenicities and diverse mycotoxin profiles; or (3) increased sexual reproduction that results in continuous distributions of toxigenicity. Population shifts that result in changes in ploidy or nuclear DNA composition (homokaryon versus heterokaryon) may have immediate effects on fitness and the rate of adaptation in subsequent fungal generations. We found that qualitative and quantitative variation in toxin phenotypes is possible through genetic recombination via crossing over within the aflatoxin gene cluster between divergent lineages in populations and between closely related species. During adaptation, specific toxin genotypes may be favored and swept to fixation or be subjected to drift and frequency-dependent selection in nature. Results from mating experiments in the laboratory indicate that fertility differences among lineages may be driving genetic and functional diversity. Differences in fertility may be the result of female sterility, changes in heterokaryotic state, DNA methylation, or other epigenetic modifications. The extent to which these processes influence aflatoxigenesis is largely unknown, but is critical to understand for both fundamental and practical applications, such as biological control. Our work shows that a combination of population genetic processes, especially asexual/sexual reproduction and balancing selection coupled with ecological factors, may influence toxin diversity in these agriculturally important fungi.Species in Aspergillus section Flavi commonly infect agricultural staples such as corn, peanuts, cottonseed, and tree nuts and produce an array of mycotoxins, the most potent of which is aflatoxin. Aspergillus flavus is the dominant aflatoxin-producing species in the majority of crops. Populations of aflatoxin-producing fungi may shift in response to: (1) clonal amplification that results from strong directional selection acting on a nontoxin- or toxin-producing trait; (2) disruptive selection that maintains a balance of extreme toxigenicities and diverse mycotoxin profiles; or (3) increased sexual reproduction that results in continuous distributions of toxigenicity. Population shifts that result in changes in ploidy or nuclear DNA composition (homokaryon versus heterokaryon) may have immediate effects on fitness and the rate of adaptation in subsequent fungal generations. We found that qualitative and quantitative variation in toxin phenotypes is possible through genetic recombination via crossing over within the aflatoxin gene cluster between divergent lineages in populations and between closely related species. During adaptation, specific toxin genotypes may be favored and swept to fixation or be subjected to drift and frequency-dependent selection in nature. Results from mating experiments in the laboratory indicate that fertility differences among lineages may be driving genetic and functional diversity. Differences in fertility may be the result of female sterility, changes in heterokaryotic state, DNA methylation, or other epigenetic modifications. The extent to which these processes influence aflatoxigenesis is largely unknown, but is critical to understand for both fundamental and practical applications, such as biological control. Our work shows that a combination of population genetic processes, especially asexual/sexual reproduction and balancing selection coupled with ecological factors, may influence toxin diversity in these agriculturally important fungi.