| Summary: | The aim of this study was to compare the performance of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), phenotypic and molecular methods for the identification of <i>Fusarium</i> species complexes isolated from clinical cases in the State of Sao Paulo (Brazil) between the years 2001 and 2017. Sequencing of ITS region of ribosomal DNA and elongation factor 1 alpha gene (ET1α) were used as reference method in the analysis of a total of 108 <i>Fusarium</i> spp. clinical strains isolated from human hosts with superficial and systemic infections. Agreement between MALDI-TOF-MS and molecular data was observed for 97 out of 108 clinical isolates (89.8%), whereas five (4.6%) and six (5.5%) clinical isolates were misidentified and were not identified by MALDI-TOF MS, respectively. ITS region sequences and MALDI-TOF MS mass spectra identified and grouped correctly most of <i>Fusarium</i> clinical isolates at species complex level. This investigation highlights the potential of MALDI-TOF MS technique as a fast and cost-efficient alternative for clinical <i>Fusarium</i> identification. However, MALDI-TOF MS requires a more accurate and larger database. This work is the first comprehensive report for <i>Fusarium</i> population, based on phenotypic analyses, proteomic profile by MALDI-TOF and phylogenetic analyses of <i>Fusarium</i> species complexes isolated from clinical cases in the State of Sao Paulo, Brazil.
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