A simple polymerase chain reaction (PCR) method for identification of the four species of Saccharomyces sensu stricto was developed. The method is based on amplification of the RPL2 region using three primers designed to detect the structural differences between the RPL2 flanking regions of S. cerevisiae and S. bayanus. A 1.1-kb DNA fragment was amplified in both S. cerevisiae and S. paradoxus, although the amount of amplified DNA in S. paradoxus was small compared to that in S. cerevisiae. A 1.9-kb DNA fragment was amplified specifically in S. bayanus. Both 1.1-kb and 1.9-kb DNA fragments were amplified in S. pastorianus, which is thought to be a hybrid species of S. cerevisiae and S. bayanus. Thus, using the three primers, the four species of Saccharomyces sensu stricto could be easily distinguished by the size and amount of the PCR product.