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• All three rhizome segments of different ages in herbaceous peony were suitable for propagation. • Chilling treatment of herbaceous peony rhizomes resulted in a higher shoot emergence rate and height. • Six to eight weeks of chilling were sufficient to break the dormancy of rhizome in herbaceous peony. • Carbohydrate, soluble protein, antioxidant enzyme and hormone changes after chilling treatment regulated the dormancy status of rhizome. The asexual propagation of herbaceous peony is common, and its efficiency can be improved by using rhizomes. However, rhizomes and rhizome buds of herbaceous peony require a low temperature treatment to break dormancy to allow for normal growth. ‘Fen Yu Nu’ (an early-flowering cultivar) and ‘Tuan Ye Hong’ (a mid-flowering cultivar), which showed high rates of shoot emergence from dormant rhizomes, were used in this study. Rhizomes segments of three ages (1- to 3-year-old) were chilled at 4 °C for 0, 2, 4, 6 and 8 weeks. Shoot emergence and growth were evaluated after 1 and 2 months in the greenhouse, while changes in nutritional compounds, enzymes, and hormones were measured during chilling and early growth to explore dormancy-release mechanisms. It was found that the three rhizome segments of different ages were suitable for propagation. Six weeks of chilling broke dormancy of ‘Fen Yu Nu’ rhizomes (eight weeks in the case of ‘Tuan Ye Hong’). The most pronounced differences in shoot emergence among treatments were observed after one month of growth, highlighting this as the key time point for assessing dormancy release efficiency. Carbohydrates, enzymes and hormones varied in different cultivars, changes that may affect the rhizome dormancy. An appropriate chilling period can effectively break rhizome dormancy and result in a higher shoot emergence rate and height, accompanied by several physiological and biochemical changes that regulate the emergence from dormancy.