The gas chromatography of hexane extracts from ascocarps of Tuber maculatum (mostly the first report), T. aestivum/unicantum, T. borchii, T. melanosporum and Tirmania nivea dominantly showed palmitic, stearic, oleic and linoleic acids followed by traces of polyunsaturated fatty acids. The fatty acid content varied from ca. 8–61 mg g− 1, dry-weight-basis with species with highest for T. maculatum. Polyunsaturated fatty acids contributions varied from ca. 42–59%. The dominant fatty acid varied with the species. A comparison with existing reports on same species cultivated in different regions showed differences in contributions by saturated, monounsaturated and polyunsaturated fatty acids as well as dominant fatty acids detected. Lesser explored species such as T. borchii, T. maculatum call for further research. This is a preliminary study that indicates fatty acid composition as a potential tool for distinction like aroma between truffle species and geographies of cultivation. This forms the basis for further studies in different species and regions.