ABSTRACT Trichosporon species are relevant etiologic agents of hospital-acquired infections. High mortality rates are attributed to Trichosporon deep-seated infections in immunocompromised individuals, making fast and accurate species identification relevant for hastening the discovery of best-targeted therapy. Recently, Trichosporon taxonomy has been reassessed, and three genera have been proposed for the pathogenic species: Trichosporon , Cutaneotrichosporon , and Apiotrichum . Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) has replaced old phenotypic methods for microorganism identification in clinical laboratories, but spectral profile databases have to be evaluated and improved for optimal species identification performance. Vitek MS (bioMérieux) is one of the commercially available MALDI-TOF MS platforms for pathogen identification, and its spectral profile databases remain poorly evaluated for Trichosporon , Cutaneotrichosporon , and Apiotrichum species identification. We herein evaluated and improved Vitek MS for the identification of the main clinical relevant species of Trichosporon , Cutaneotrichosporon , and Apiotrichum using a large set of strains and isolates belonging to different yeast collections in Brazil and France.