Ochratoxin A (OA) is a mycotoxin that has been found in coffee beans and coffee beverages. Its toxicological profile includes carcinogenicity, nephrotoxicity, and immunotoxicity. Aspergillus ochraceus is the major species responsible for OA production in Brazilian coffee beans. The genetic relationships among 25 A. ochraceus strains collected from Brazilian coffee-bean samples were determined based on RAPD and internal transcribed spacer (ITS) sequence data. The isolates were resolved into 2 distinct groups, one with 4 strains (group A) and the other with 21 strains (group B). Specific nucleotide variations characterizing group A and B were found for both ITS1 and ITS2 regions. Group B is a new group proposed here to accommodate the majority of the Brazilian isolates. Each group was found to contain both toxigenic and nontoxigenic strains, indicating that there is no association between molecular genotypes and the ability to produce OA.