Aims: To evaluate a new platform for yeast differentiation based on histone promoter regions. Methods and Results: The histone gene amino acid sequences of a wide phylogenetic range of organisms were aligned, and primers designed that were capable of amplifying the divergent promoters of the H3-H4 and H2a-H2b loci from yeast. Analysis indicated that the promoter regions were variable in length between species and represented rapidly changing sequences flanked by highly conserved sequences. Conclusions: The histone promoter regions in yeast provide an excellent locus for the rapid and accurate identification of yeast species. Significance and Impact of the Study: This study describes an alternative platform to the ribosomal internal transcribed spacer (ITS) sequences for the identification of yeast species.