Polymorphisms in enzymatically amplified ribosomal DNA (rDNA) were examined in 18 strains of Saccharomyces. Restriction patterns generated from the region spanning the internal transcribed spacers (ITS) and the 5.8S rDNA produced two clusters corresponding to S. bayanus and S. cerevisiae. The type culture of S. carlsbergensis (ATCC 76529), which could not be separated from the S. cerevisiae group by small subunit (SSU) rDNA patterns, showed a ScrfI profile that was distinct from all the other strains. The type culture of S. intermedius var. turicensis (ATCC 76519) is assigned to S. bayanus on the basis of the combined results of SSU and ITS restriction analyses. S. kluyveri occurred at a separate branch of the distance tree and is unrelated to any of the strains. Results were in general agreement with reported DNA homologies and are discussed in relation to other molecular and genetic data.