Accurate species identification of the Sporothrix schenckii complex is essential, since identification based only on phenotypic characteristics is often inconclusive due to phenotypic variability within the species. We used matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) for species identification of 70 environmental and clinical isolates of the Sporothrix complex. A reference database was established for MALDI-TOF MS-based species identification according to minor adjustments in the manufacturer's guidelines. The MALDI-TOF MS clearly distinguished strains of Sporothrix brasiliensis, Sporothrix globosa, Sporothrix mexicana, S. schenckii, Sporothrix luriei and Sporothrix pallida, enabling identification of all isolates at the species level, as confirmed by partial calmodulin gene sequence analyses. The present methodology is simple, reliable, rapid and highly suitable for routine identification in clinical mycology laboratories and culture collections, particularly for updating and reclassifying of deposited Sporothrix isolates.The authors wish to thank the following international researchers for generously contributing strains to this study: Conchita Torrielo (EH194, EH252, EH253); Myrtha Arango (04015, 11029, 010221, 10036, 03017, 03022, 12013, 03003, 14879); and Masako Kawasaki (KMU975). Financial support was provided by FAPERJ/Rio de Janeiro, Brazil (grant proc. E-26/110.619/2012) and PAPES VI-Fiocruz/CNPq (Proc. 407693/2012-2) R. M. Z-O. is supported in part by CNPq 304976/2013-0 and FAPERJ E-26/103.157/2011. M. M. E. O. was supported by a grant from CAPES 2445/11-5 and PNPD/CAPES-Fiocruz/Pesquisa Clinica em Doencas Infecciosas. M. M. E. O., C. S. and N. L. thank the FCT Strategic Project PEst-OE/EQB/LA0023/2013 and BioHealth-Biotechnology and Bioengineering approaches to improve health quality, Ref. NORTE-07-0124-FEDER-000027" co-funded by the Programa Operacional Regional do Norte (ON.2 - O Novo Norte), QREN, FEDER. Automated sequencing was done using the genomic platform/DNA sequencing platform at the Fundacao Oswaldo Cruz-PDTIS/FIOCRUZ (RPT01A), Brazil
