It is reported that heat stress could induce the biosynthesis of ganoderic acids and enhance the production of G. lingzhi polysaccharides. However, heat stress inhibits mycelium growth and reduces hyphal branching, resulting in a significant reduction in mycelial dry weight. Tandem mass tag-based (TMT) comparative proteomic analysis was performed to explore the potential mechanisms associated with thermotolerance in G. lingzhi in this study.