[Background] Auxotrophy is a widely applied biomarker in production and scientific research, but there is no report on Ganoderma lucidum. [Objective] This study was conducted to provide parent strains and technique supports for genetics research, cross breeding and spawn identification of Ganoderma lucidum. [Methods] The uracil auxotrophic mutant strains were obtained by UV mutagenesis of protoplast. The randomly selected uracil auxotrophic monokaryon strains were used as parents, and crossed with each other by Monkaryon-monkaryon mating to establish uracil auxotrophic dikaryons. [Results] eight uracil auxotrophic mutant strains were obtained by UV mutagenesis of protoplast. seven uracil auxotrophic dikaryon strains were obtained by Monkaryon-monkaryon crossing. [Conclusion] The testing results showed these auxotrophic strains restored their growth on PDA medium with uracil. The uracil auxotrophic dikaryon strains would be used as a tool for genetic transformation study and breeding.