Ochratoxin A (OA) is a nephrotoxic and carcinogenic mycotoxin that has been found in cereal and food commodities. Currently, Aspergillus carbonarius, A. niger and A. ochraceus have been recognized as the species responsible for OA in coffee beans. With the aim of developing multiplex-PCR for detection of these species in coffee bean samples, we first developed specific primers for A. niger detection. The primer designed (OPX7(372)) provided an amplicon of 372 pb in all A. niger stricto sensu isolates. The PCR assay developed for A. niger identification in pure culture was also successfully used for detecting this species in coffee beans. No cross-reaction was observed using DNA from coffee beans inoculated with closely related black aspergilli species. A multiplex-PCR method for detection of A. carbonarius, A. niger and A. ochraceus species in coffee beans was developed. From a single assay this method detected the amplicons of 809, 372, and 260 pb that represent the three most ochratoxigenic species found in coffee bean samples, i.e., A. carbonarius, A. niger and A. ochraceus, respectively.