A study of DNA reassociation kinetics and electrophoretic karyotypes of several strains classified as Saccharomyces castellii, Saccharomyces dairensis, and Pachytichospora transvaalensis strains revealed that the group included members of at least five distinct species. Two of the four strains classified as P. transvaalensis exhibited very high levels of DNA sequence homology with the type strain of S. castellii, while the type strain and another strain demonstrated intermediate levels of relatedness to the type strains of the unrelated taxa S. castellii and S. dairensis. Five strains that were classified as S. diarensis strains were not related to the type strains of the taxa studied. Two of these strains exhibited intermediate relatedness (65% base sequence homology), while another pair exhibited a level of DNA reassociation of 97%. The remaining strain was distinct from all the other strains studied on the basis of both DNA base sequences and electrophoretic karyotype data.