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Intentional substitution of food ingredients and misleading or false declarations of food products can lead to large economic damage in the food sector. An example is the truffle, an edible mushroom, with different species having a similar morphology. This makes it difficult to distinguish, for example, the high-quality black Périgord truffle from Europe and the lower quality black truffle from China. To detect and prevent potential food fraud and to be able to carry out quality assurance measures, the demand for suitable, fast and cost-effective examination methods for individual foodstuffs is increasing. In this article we present a method based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), which can differentiate and identify commercial truffles. For this purpose, highly specific mass spectrometric fingerprints of 73 truffle tubers from eight different species were generated. These were checked and compared under the criteria of a score evaluation, their protein spectra, and cluster formation. Standard laboratory equipment was used for this purpose, so that apart from the MALDI-TOF no expensive equipment or reagents were required. The study shows that the MALDI-TOF MS method can distinguish and identify Tuber spp. reliably, quickly and efficiently and can therefore be used for quality assurance and fraud control in the truffle market., (© 2020 John Wiley & Sons Ltd.)