In this study, we identified a total of 33 wine yeast species and strains using the restriction patterns generated from the region spanning the internal transcribed spacers (ITS 1 and 2) and the 5.8S rRNA gene. Polymerase chain reaction (PCR) products of this rDNA region showed a high length variation for the different species. The size of the PCR products and the restriction analyses with three restriction endonucleases (HinfI, CfoI, and HaeIII) yielded a specific restriction pattern for each species with the exception of the corresponding anamorph and teleomorph states, which presented identical patterns. This method was applied to analyze the diversity of wine yeast species during spontaneous wine fermentation.