Truffles are appreciated as food all over the world because of their extraordinary aroma. However, quantities are limited and successful cultivation in plantations is very labor-intensive and expensive, or even impossible for some species. These factors make truffles a very valuable food, which is why it is particularly rewarding and tempting to declare inferior species of truffles as more expensive species and thereby counterfeit them. The various species differ in their aroma and thus in their culinary value, but the adulterations cannot be detected on the basis of pure morphology. For this reason, the objective of the present study was to develop a non-targeted lipidomics approach using ion mobility spectrometry-mass spectrometry to distinguish between the white truffle species Tuber magnatum and T. borchii as well as the black truffle species T. melanosporum, T. aestivum and T. indicum. Several hundred features were detected, which were present in significantly different concentrations in the studied truffle species. The most important of them were identified using MS/MS spectra and collision cross section (CCS) values. Some compounds were detected whose CCS values have not yet been published and may facilitate identification by other researchers in the future. Just a few marker substances are sufficient to be able to distinguish both black and white truffle species with 100% accuracy. These results can be used for the development of rapid tests, which in the best case will allow truffle analysis directly on-site.