Abstract: In this paper,the orthogonal design was used to optimize DDRT-PCR amplification system on Racomitrium japonicum in five levels of six factors(Mg²⁺,dNTP,anchor primer,random primer,DNA template,Taq DNA polymerase) respectively.We established a most suitable DDRT-PCR system for R.japonicum,which contains Mg²⁺ 2.25 mmol/L,dNTP 0.4 mmol/L,anchor primer 1.0 μmol/L,random primer 0.7 μmol/L,DNA template 1.6 μL,and Taq DNA polymerase 2.5 U in 20 μL reaction system.At the last,the optimal annealing temperature for DDRT-PCR reaction was proposed by gradient PCR and it’s 45.4℃.The result provided an important reference for the selecting and cloning of drought-resistant genes of R.japonicum.