AIM: To develop the matrix-assisted laser desorption ionization mass spectrometry (MALDI-TOF MS) method for identification of Fusarium species within Fusarium fujikuroi complex for use in clinical microbiology laboratories. MATERIALS & METHODS: A total of 24 reference and 60 clinical and environmental isolates belonging to the F. fujikuroi complex were subjected to MALDI-TOF MS identification. Protein extracts of Fusarium isolates were obtained using formic acid extraction. Multilocus sequence analysis was used as a gold standard. RESULTS: The MALDI-TOF MS Biotyper correctly identified 93.6% of the strains down to the species level, while the remaining isolates (6.4%) were identified at the genus level when using scores of >/= 2.0 as cut-off values. Correct identification was obtained despite large intraspecific heterogeneities in MALDI-TOF spectra. CONCLUSION: MALDI-TOF MS is a rapid identification tool for the recognition of species within F. fujikuroi complex, provided a database is available