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Background: A Pyrosequencing assay has been used in identification of fungal species such as Candida or Aspergillus and diagnosis of pathogenic bacteria such as Helicobacter pylori but there has been no report on successful isolation and identification of Malassezia yeasts using the pyrosequencing method. Objective: Examine the applicability and plausibility of the pyrosequencing method in identification of the Malassezia species. Methods: At internal transcribed spacer (ITS) sites 1 and 2, three primers were developed using Pyrosequencing Assay Design Software (Biotage AB). Pyrosequencing was performed on 11 standard strains and 83 genomic DNA samples obtained from 66 healthy controls aged from 1 to 80. Results: The eleven Malassezia standard species and 83 genomic DNA samples were successfully identified using the pyrosequencing assay. Conclusion: The pyrosequencing method is a new tool for analysis of Malassezia yeasts, and its precision and rapidity suggests its clinical applicability.