AbstractThe PfURA3 gene, which encodes orotidine‐5′‐phosphate decarboxylase, of osmotolerant yeast Pichia farinosa NFRI 3621, was cloned by complementation of the ura3 mutation of Saccharomyces cerevisiae. The nucleotide sequence of the PfURA3 gene and its deduced amino acid sequence indicated that the gene encodes a protein (PfUra3p) of 267 amino acids. Pulsed‐field gel electrophoresis and subsequent Southern blot analysis showed that the genome of P. farinosa NFRI 3621 consisted of seven chromosomes, each approximately 1.1–2.2 Mb in size (11.8 Mb in total) and that PfURA3 was located on chromosome V. Pichia sorbitophila is considered as a synonym of P. farinosa. The genome of P. sorbitophila IFO10021 may consist of 12 chromosomes, each approximately 1.2–2.2 Mb in size. P. sorbitophila has two copies of URA3 genes, termed PsURA3 and PsURA30, which were located on chromosome VIII and III, respectively. The difference between PfURA3 and PsURA3 was only two amino acid substitutions, whereas that between PsURA3 and PsURA30 was six amino acid substitutions and the deletion of the C‐terminal amino acid by a stop codon insertion. The sequences of PfURA3, PsURA3 and PsURA30 have been deposited in the DDBJ data library under Accession Nos AB071417, AB109042 and AB109043, respectively. Copyright © 2003 John Wiley & Sons, Ltd.