Abstract: In order to screen suitable media and transplanting substrate for the tissue culture and rapid propagation of Cymbidium hybrid ‘Zhongtou Ziyanshi’, thereby obtaining genetically stable tissue-cultured seedlings and effectively addressing the issue of leaf transparency variegation degradation during the tissue culture process. The effects of different plant growth regulator combinations on rhizome induction, proliferation, differentiation, strengthen seedlings and rooting were investigated. Additionally, the impact of various transplant substrates on the survival and growth of tissue-cultured seedlings was compared. Furthermore, the genetic stability of the tissue-cultured seedlings was assessed using EST-SSR molecular marker primers. The results showed that the highest rhizome induction rate was achieved in the MS1 medium supplemented with 2.0 mg·L⁻¹ 6-BA and 0.2 mg·L⁻¹ NAA, while the MS2 medium supplemented with the combination of 0.8 mg·L⁻¹ KT and 0.5 mg·L⁻¹ NAA demonstrated favorable effects on rhizome proliferation and differentiation, with a leaf transparency variegation retention rate 94.54%. The MS3 medium supplemented with the combination of 0.1～0.2 mg·L⁻¹ 6-BA and 1.0 mg·L⁻¹ NAA were found to be more suitable for seedling strengthening and rooting, and a mixture of fine bark and perlite in a 4∶1 ratio was identified as an effective transplanting substrate with higher survival rate. The tissue-cultured seedlings confirmed their strong genetic stability, with no DNA-level variations detected.