Sequences of two internal transcribed spacer regions between 18S and 28S rRNA for recently described yeasts species, Saccharomyces kunashirensis, S. martiniae, S. rosinii, and S. transvaalensis, were determined to assess their phylogenetic relationship to the other Saccharomyces species. In the two phylogenetic trees constructed by the neighbor-joining method, independent branches reflected that delimitation of the four new species was valid.