Aims To use genome-wide single nucleotide polymorphisms (total SNPs) to develop a molecular method for distinguishing Aspergillus flavus and Aspergillus oryzae. Methods and Results Thirteen A. flavus and eleven A. oryzae genome sequences were obtained from the National Center for Biotechnology Information. These sequences were analysed by Mauve, a multiple-genome alignment program, to extract total SNPs between isolates of A. flavus, A. oryzae, or the two species. Averages of total SNPs of A. flavus isolates belonging to the same sclerotial morphotype (L-type = 178 952 +/- 14 033; S-type = 133 188 +/- 16 430) and A. oryzae isolates (152 336 +/- 49 124) were consistently lower than those between the morphotypes and between the two species. Averages of total SNPs for L-type vs S-type (300 116 +/- 1562) and S-type A. flavus vs A. oryzae (301 797 +/- 4123) were similar but were 36% greater than that of L-type A. flavus vs A. oryzae (226 240 +/- 10 779). Based on the devised criterion, ATCC 12892, Aspergillus oryzae (Ahlburg) Cohn, which had an averaged total SNPs 10-fold greater than that of other A. oryzae isolates, was determined to be close to Aspergillus parasiticus. Atoxigenic A. flavus field isolates, WRRL1519 and NRRL35739, were shown to more closely resemble A. oryzae than toxigenic L-type A. flavus. Biocontrol strains AF36 and K49 were genetically close to toxigenic L-type A. flavus. NRRL21882, the active agent of the commercialized biocontrol product Afla-Guard (R) GR, was genetically distant from all other A. flavus isolates. Conclusions The close genetic relatedness between A. flavus and A. oryzae was confirmed and the evolutionary origins of atoxigenic A. flavus biocontrol strains were revealed. Significance and Impact of the Study The study provides a greater understanding of genome similarity and dissimilarity between A. flavus and A. oryzae. The method can be an auxiliary technique for identifying A. flavus, A. oryzae.