Ganoderma lucidum is one of the most commonly used mushrooms in traditional Chinese medicine, with significant immunomodulatory and antitumor effects. Triterpenoids are deemed to be the main bioactive components in G. lucidum. In this study, high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (MS) and high-performance liquid chromatography-MS/MS methods were used to identify and quantify, respectively, the main triterpenoids in G. lucidum obtained from different sources. The full MS scan with a daughter ion scan was used to identify a variety of potential derivatives of triterpenoids in the mushroom. Multiple reaction monitoring was performed with transitions of m/z-515.2 → 285.1 (quantifier) and 515.2 → 497.2 (qualifier)-to determine the amount of ganoderic acid A. The developed methods were successfully used to analyze 8 G. lucidum samples from the wild and from cultivated and commercial sources. The results show that the content and composition of triterpenoids differed significantly among these samples. A total of 29 triterpenoids were explicitly or tentatively identified in the wild sample, whereas 23 triterpenoids were found in the cultured sample and 17 in the commercial sample. The results of this study provide a foundation for further research on chemical constituents of, identification of metabolites in, and quality control of the G. lucidum mushroom.