The fungal phylum Ascomycota comprises a large proportion of species with no known sexual stage, despite high genetic variability in field populations. One such asexual species, Aspergillus parasiticus, is a potent producer of carcinogenic and hepatotoxic aflatoxins, polyketide-derived secondary metabolites that contaminate a wide variety of agricultural crops. In this study, individuals of A. parasiticus from a population showing an evolutionary history of recombination were examined for sexual reproduction. Crosses between strains with opposite mating-type genes MAT1-1 and MAT1-2 resulted in the development of ascospore-bearing ascocarps embedded within stromata. Sexually compatible strains belonged to different vegetative compatibility groups. Recombination through the independent assortment of chromosomes 3 and 6 was detected using loci for mating type, aflatoxin gene cluster, and a protein-encoding gene. Our discovery of the sexual stage in A. parasiticus has important implications for current biological control strategies using nontoxigenic strains to reduce aflatoxin contamination in crops.