Restriction digestion analysis of the acyl transferase (AT) domain sequences of a polyketide synthase (PKS) gene was tested as a rapid method to identify isolates of Aspergillus tubingensis from grapes. Restriction endonuclease digestion of PKS products using the endonucleases BccI, HaeIII, HpaII, MboI and TaqI distinguished five types of restriction fragment length polymorphism (RFLP). Ochratoxigenic isolates were only identified within RFLP-types I and III. The RFLP assay is proposed as a rapid and easy method to identify A. tubingensis isolates from grapes. Amino acid sequences of AT domains from representative A. tubingensis isolates of the RFLP types obtained were aligned and analysed using phylogenetic methods. A comparison was also made with reference strains of Aspergillus section Nigri. Most of the A. tubingensis strains clustered into two distinct groups Gr1 and Gr2 with the exception of two isolates that remained unclustered. These results support the intraspeficific variability within A. tubingensis species reported using other techniques.