A comparison of lipid content and fatty acid (FA) composition between Tuber fermentation mycelia and natural fruiting bodies indicates that the lipid content in Tuber fermentation mycelia is higher than that in fruiting bodies. Unsaturated FAs (particularly linoleic acid and oleic acid) were the predominant constituents in total FAs in both Tuber fermentation mycelia and fruiting bodies. A total of 23 FAs, including arachidonic, eicosapentaenoic, docosahexaenoic, and γ-linolenic acids, were first identified in the Tuber species. A hierarchical clustering analysis showed that the FA profile of fermentation mycelia was quite similar, regardless of Tuber species. However, the FA profile of the fruiting bodies was significantly influenced by its species and habitat environments. Interestingly, the FA profile of the Tuber indicum and Tuber aestivum fruiting bodies was nearly identical to that of the Tuber fermentation mycelia, which partially confirms the similarity between the Tuber fermentation mycelia and the fruiting bodies.