Single nucleotide polymorphisms (SNPs) of genome sequences of eight Aspergillus flavus and seven Aspergillus oryzae strains were extracted with Mauve, a multiple‐genome alignment programme. A phylogenetic analysis with sequences comprised of concatenated total SNPs by the unweighted pair group method with arithmetic mean (UPGMA) of MAFFT adequately separated them into three groups, A. flavus S‐morphotype, A. flavus L‐morphotype and A. oryzae. Divergence time inferred for A. flavus NRRL21882, the active agent of the biocontrol product Afla‐Guard®, and S‐morphotype was about 5·1 mya. Another biocontrol strain, A. flavus AF36, diverged from aflatoxigenic L‐morphotype about 2·6–3·0 mya. Despite the close relatedness of A. oryzae to A. flavus, A. oryzae strains likely evolved from aflatoxigenic Aspergillus aflatoxiformans (=A. parvisclerotigenus). A survey of A. flavus populations implies that prior Afla‐Guard® applications are associated with prevalence of NRRL21882‐type isolates in Mississippi fields. In addition, a few NRRL21882 relatives were identified. A. flavus Og0222, a biocontrol ingredient of Aflasafe™, was verified as a NRRL21882‐type strain, having identical sequence breakpoints that led to deletion of aflatoxin and cyclopiazonic acid gene clusters. A similar UPGMA analysis suggests that the occurrence of NRRL21882‐type strains is a more recent event. Significance and Impact of the Study: Toxigenic Aspergillus flavus infects crops and produces aflatoxin, which is a great threat to human and animal health. On the contrary, nonaflatoxigenic A. flavus is currently being used as a biocontrol agent. Their close relative, Aspergillus oryzae, has been widely used in food fermentation for centuries. This study provides a better understanding of genome similarity and dissimilarity between these two species. It also reveals the evolutionary origin of a particular biocontrol strain, NRRL21882, and the presence of its close relatives in field A. flavus populations. Information obtained should assist selection and characterization of nonaflatoxigenic A. flavus strains for effective biocontrol.