Mitochondrial DNA restriction fragment length polymorphisms (RFLPs) are described for 64 isolates, representing 11 species of Aspergillus section Flavi. Mitochondrial DNA haplotypes were identified following digestion of total cellular DNA with the restriction enzymes HaeIII, AseI, or DraI. In general, isolates of the same species possessed identical mitochondrial DNA haplotypes. Three haplotypes were found in multiple, closely related species: one in A. flavus, A. oryzae, and A. subolivaceus; a second in A. parasiticus and A. sojae; and a third in A. tamarii and A. flavofurcatis. Four distinct haplotypes were each associated with a single species: A. nomius, A. avenaceus, A. leporis, and A. zonatus. Mitochondrial DNA haplotypes complement traditional morphological and growth criteria in making taxonomic decisions within Aspergillus section Flavi.