Poor rooting and acclimatization are two main problems related to the micropropagation of tree peony. In view of the negative N⁶-benzyladenine (BA) carryover effect on rooting and acclimatization reported in many plant species, this study aimed at optimizing the micropropagation of tree peony (Paeonia × lemoinei ‘High Noon’) by substituting BA with meta-topolin (mT; used alone or in combination with NAA and GA₃) during shoot multiplication to enhance rooting and acclimatization. The combinations of mT or BA with GA₃ significantly promoted the shoot multiplication and growth compared with cytokinin alone. The highest number of shoots in mT and BA treatments were obtained with 5 μM mT + 1.5 μM GA₃ (2.7) and 5 μM BA + 1.5 μM GA₃ μM (2.7), respectively. During rooting and acclimatization, the shoots produced on mT-supplemented medium showed significantly higher rooting percentage and survival rate than those produced on BA-supplemented medium. High rooting (62.0%) and survival rate (91.2% ) were observed in shoots pretreated with 5 μM mT + 1.5 μM GA₃, which were substantially higher than those observed in shoots pretreated with 5 μM BA + 1.5 μM GA₃ (rooting 47.0%, survival 83.5%). Furthermore, the absence of genetic variations was confirmed in micropropagated plants using 20 simple sequence repeat (SSR) markers. Therefore, the combined use of mT with GA₃ during shoot multiplication may provide a method to ensure the efficient micropropagation of tree peony.