The internal transcribed spacer(ITS) region of nuclear DNA is one of the most used locus for yeast identification with growing sequence data deposited in public-access databases.This study extensively evaluated the feasibility of ITS sequence analysis for systemic classification of yeast isolates.The ITS regions of 623 yeast strains deposited in CICIM-CU were amplified by PCR and sequenced,the sequences were compared to reference data available at the GenBank database by using BLAST.Most of fungal isolates(93.3%) were assigned to species level successfully.40 strains(6.4%) were only can be assigned to genus level.2 strains cannot be identified based on ITS sequences analysis.This study shown ITS sequences were sufficiently sensitive for identifying most of yeasts,it can be powerfully and ordinarily used as first-round barcoding gene to systemic identification of bulk yeast isolates.