The ratios of stable carbon isotopes ((13)C/(12)C) of ganoderma fruiting body, ganoderma spore, ganoderma spore lipid (GSL) and individual fatty acids in GSL were determined by gas chromatography-stable isotope ratio mass spectrometry and elemental analysis-stable isotope ratio mass spectrometry. These values fall into a range from -26.9 to -23.3 per thousand, suggesting that the cut log as the Ganoderma-cultivated substrate in Fujian, China, may belong to C3 plants. Eighteen fatty acids were identified and their abundances measured by gas chromatography-mass spectrometry in the six GSL samples with C(16:0), C(18:0), C(18:1) and C(18:2) as major constituents, and C(16:1) is evidently enriched compared with the other edible vegetable oils. On the basis of the compositions of fatty acids and stable carbon isotopes in GSL, we have developed a novel method to detect the adulteration of GSL products with cheaper edible vegetable oils. An example of ideal blending between GSL and C4 or C3 vegetable oil is further provided to expound the discrimination procedures and corresponding sensitive indicators. Simultaneously, the carbon isotope fractionation in the biosynthesis of individual fatty acids was observed, revealing that the formation of C(18:0) from C(16:0) in ganodema spores had no conspicuous (13)C enrichment of +0.4 per thousand for Ganoderma sinensis spore and +0.1 per thousand for G. lucidum spore; the desaturation of C(18:0) to C(18:1) resulted in a distinct (13)C depletion of -1.4 per thousand for G. sinensis spore and -0.9 per thousand for G. lucidum spore; and the next desaturation from C(18:1) to C(18:2) displayed no evident (13)C fractionation of -0.1 per thousand for G. sinensis spore and -0.2 per thousand for G. lucidum spore.