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<i>Ganoderma lingzhi</i> is an important medicinal fungus, and it is particularly important to select strains with high yields and active substance contents. In this study, protoplasts of <i>G. lingzhi</i> were thermally inactivated to destroy intracellular enzyme proteins and preserve DNA. The DNA of <i>G. resinaceum</i> was damaged by ultraviolet (UV) radiation, and other components of the protoplasm except DNA were preserved. Then, the protoplast was induced using polyethylene glycol (PEG) for fusion. The results showed that the optimal thermal inactivation conditions for <i>G. lingzhi</i> were 30 min in a 45 °C water bath, and the optimal UV inactivation conditions for <i>G. resinaceum</i> were 70 s of irradiation using a 20 W UV lamp at a vertical distance of 15 cm. Antagonistic tests, internal transcribed space (ITS) and mitochondrial DNA identification, intersimple sequence repeat (ISSR) molecular markers and morphology were used to distinguish the parents from the fusants. Four true fusants were obtained, and the yield was 2.5%. The fruiting body yield of the fusants was significantly higher than that of <i>G. lingzhi</i>, and the polysaccharide and triterpene contents of the RAD-64 fusant were significantly higher than those of <i>G. lingzhi</i>. The results presented in this paper show that protoplast fusion technology can effectively improve <i>G. lingzhi</i> varieties and support the breeding of new varieties.