The incidence of invasive fungal infection has increased significantly. A majority of the infections is caused by yeast. Clinically important yeast show species-specific differences in susceptibility to antifungal agents therefore rapid and accurate identification of the pathogen is essential. We aimed to validate pyrosequencing of 40 nucleotides in the internal transcribed spacer 2 (ITS2) for species identification of yeast. Amplification of ITS2 and pyrosequencing of targeted region were performed in 940 clinical isolates of yeast. A local database containing the 40 nucleotide ITS2 sequences of 33 species of medically important yeast was generated using published sequences of type strains. The sequencing results were searched against the local database using the BLAST algorithm to identify the species of yeast. The length of sequences obtained from pyrosequencing averaged between 40-61 nucleotides. Pyrosequencing identified 940 clinical isolates of yeast down to 14 species level, whereby 931 isolates belonged to genus Candida (11 species), four of Saccharomyces cerevisiae, three of Malassezia pachydermatis and two of Rhodotorula mucilaginosa. In addition, intraspecies specific sequence variations in Candida albicans and Candida glabrata were detected. Pyrosequencing of 40 nucleotides in ITS2 is reliable for species identification of yeast. This methodology can contribute to the high quality management of patients with fungal infections.