Fusarium spp. have recently emerged as significant human pathogens. Identification of these species is important, both for epidemiological purposes and for patient management, but conventional identification based on morphological traits is hindered by major phenotypic polymorphism. In this study, 62 strains, or isolates, belonging to nine Fusarium species were subjected to both molecular identification TEF1 gene sequencing and matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) analysis. Following stringent standardization, the proteomic-based method appeared to be both reproducible and robust. Mass spectral analysis by comparison with a database, built in this study, of the most frequently isolated species, including Fusarium solani, Fusarium oxysporum, Fusarium verticilloides, Fusarium proliferatum and Fusarium dimerum, correctly identified 57 strains. As expected, the four species (i.e. Fusarium chlamydosporum, Fusarium equiseti, Fusarium polyphialidicum, Fusarium sacchari) not represented in the database were not identified. Results from mass spectrometry and molecular identification agreed in five of the six cases in which results from morphological and molecular identification were not in agreement. MALDI-TOF yielded results within 1 h, making it a valuable tool for identifying clinical Fusarium isolates at the species level. Uncommon species must now be added to the database. MALDI-TOF may also prove useful for identifying other clinically important moulds.