Abstract: In order to screen suitable media and transplanting substrate for the tissue culture and rapid propagation of Cymbidium hybrid ‘Zhongtou Ziyanshi’, thereby obtaining the tissue-cultured seedlings with stable genetic traits and effectively solving the problem of leaf transparency variegation degradation during the tissue culture process. The effects of different combinations of plant growth regulators on the induction, proliferation and differentiation of rhizomes and the rooting of strong seedlings were studied and compared. Additionally, the impact of various transplanting substrates on the survival rate and plant growth of tissue-cultured seedlings was compared. Furthermore, the genetic stability of the tissue-cultured seedlings was assessed by using the EST-SSR molecular marker primers. The results showed that the highest rhizome induction rate of Zhongtou Ziyanshi 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 the proliferation and differentiation of rhizomes, with the leaf transparency variegation retention rate of 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 the 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 obtained by this method had strong genetic stability, with no DNA-level variations detected.