AbstractAspergillus sojaehas long been considered a domesticated strain ofAspergillus parasiticus. This study delineated relationships among the two species and anAspergillusPWE36 isolate. Of 25 examined clustered aflatoxin genes of PWE36, 20 gene sequences were identical to those ofA. sojae, but all had variations to those ofA. parasiticus. Additionally, PWE36 developmental genes of conidiation and sclerotial formation, overall, shared higher degrees of nucleotide sequence identity withA. sojaegenes than withA. parasiticusgenes. Examination of defective cyclopiazonic acid gene clusters revealed that the PWE36 deletion pattern was identical only to those ofA. sojae. UsingA. sojaeSMF134 genome sequence as a reference, visualization of locally collinear blocks indicated that PWE36 shared higher genome sequence homologies withA. sojaethan withA. parasiticus. Phylogenetic inference based on genome-wide single nucleotide polymorphisms (SNPs) and total SNP counts showed thatA. sojaestrains formed a monophyletic clade and were clonal. Two (Argentinian and Ugandan)A. parasiticusisolates but not including an Ethiopian isolate formed a monophyletic clade, which showed thatA. parasiticuspopulation is genetically diverse and distant toA. sojae. PWE36 andA. sojaeshared a most recent common ancestor (MRCA). The estimated divergence time for PWE36 andA. sojaewas about 0.4 mya. UnlikeAspergillus oryzae, another koji mold that includes genetically diverse populations, the findings that currentA. sojaestrains formed a monophyletic group and shared the MRCA with PWE36 allowA. sojaeto be continuously treated as a species for food safety reasons.