IntroductionCodon usage bias is a prevalent phenomenon observed across various species and genes. However, the specific attributes of codon usage in the mitochondrial genome ofGanodermaspecies remain unknown.MethodsIn this study, we investigated the codon bias of 12 mitochondrial core protein-coding genes (PCGs) in 9Ganodermaspecies, including 13Ganodermastrains.ResultsThe codons of allGanodermastrains showed a preference for ending in A/T. Additionally, correlations between codon base composition and the codon adaptation index (CAI), codon bias index (CBI) and frequency of optimal codons (FOP) were identified, demonstrating the impact of base composition on codon bias. Various base bias indicators were found to vary between or withinGanodermastrains, including GC3s, the CAI, the CBI, and the FOP. The results also revealed that the mitochondrial core PCGs ofGanodermahave an average effective number of codons (ENC) lower than 35, indicating strong bias toward certain codons. Evidence from neutrality plot and PR2-bias plot analysis indicates that natural selection is a major factor affecting codon bias inGanoderma. Additionally, 11 to 22 optimal codons (ΔRSCU>0.08 and RSCU>1) were identified in 13Ganodermastrains, with GCA, AUC, and UUC being the most widely used optimal codons inGanoderma. By analyzing the combined mitochondrial sequences and relative synonymous codon usage (RSCU) values, the genetic relationships between or withinGanodermastrains were determined, indicating variations between them. Nevertheless, RSCU-based analysis illustrated the intra- and interspecies relationships of certainGanodermaspecies.DiscussionThis study deepens our insight into the synonymous codon usage characteristics, genetics, and evolution of this important fungal group.