A genomic comparison of bottom fermenting yeasts was performed by pulsed-field gel electrophoresis and Southern blot analysis with some S. cerevisiae gene probes. We confirmed that strains of bottom fermenting yeast have four chromosomes originating from S. bayanus. Since the structures of these chromosomes were recombined with S. cerevisiae chromosomes, these S. bayanus chromosomes could be differentiated from S. cerevisiae chromosomes using Southern hybridization. Our Southern hybridization results indicate that bottom fermenting yeasts have both chromosomes originating from both S. cerevisiae and S. bayanus. It was reconfirmed that top fermenting yeast should be classified as S. cerevisiae, based on the chromosomal structure. The chromosomal structure of S. pastorianus CBS1538, the type stain of S. pastorianus, was also investigated. This strain has chromosomes originating only from S. bayanus. S. carlsbergensis CBS1513 has chromosomes originating from both S. cerevisiae and S. bayanus. From these results, we contend that bottom fermenting yeasts should be classified as S. carlsbergensis.