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Aspergillus flavus, like approximately one–third of ascomycete fungi, is thought to be cosmopolitan and clonal because it has uniform asexual morphology. A. flavus produces aflatoxin on nuts, grains, and cotton, and assumptions about its life history are being used to develop strategies for its biological control. We tested the assumptions of clonality and conspecificity in a sample of 31 Australian isolates by assaying restriction site polymorphisms from 11 protein encoding genes and DNA sequences from five of those genes. A. flavus isolates fell into two reproductively isolated clades (groups I and II). The lack of concordance among gene genealogies among isolates in one of the clades (group I) was consistent with a history of recombination. Our analysis included five strains of the closely related industrial fungus A. oryzae, all of which proved to be clonally related to group I.