Members of the Fusarium fujikuroi species complex (FFSC) are commonly involved in devastating diseases of many economically important plants. They invade developing seeds and other plant tissues in the field causing significant annual losses. In addition, fungal spoilage can also affect human and animal health because some species in this group, especially F. proliferatum and F. verticillioides, are mycotoxin producers occurring in food/feed worldwide. Since morphology-based species identification is of limited value in the FFSC, the development of new methods is fundamental for accurate identification of the molds to species level. Matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) analysis of subproteomes has been applied as a promising tool for the discrimination of closely related species in many microorganisms. In the present study, MALDI-TOF MS was applied to distinguish closely related species in the FFSC and to validate the effectiveness of a standardized protocol by identifying field isolates that fulfilled the morphological characteristics of FFSC species. Forty-nine of the currently described 61 species were identified by DNA sequencing analysis and their mass spectra were included as reference in a supplementary MALDI-TOF MS database. The discriminative potential of the database was evaluated with more than 80 non-reference FFSC isolates and resulted in 94.61% of correct identifications at the species level. We demonstrate that MALDI-TOF MS is a suitable and accurate technology for the identification and differentiation of species within the FFSC as well as an innovative, time-efficient alternative to multilocus sequencing technology (MLST).