Chalcone isomerase (CHI) is a key gene in regulating the formation of yellow petals. Inner and outer petals from different Paeonia lactiflora cultivars including Jinhui, Tongyunjinyan, Hongloujinju at four growing stages (flower–bud stage, initiating bloom stage, bloom stage, wither stage) were selected as materials to study expression pattern and characteristic of CHI gene. Expression difference of CHI gene in inner and outer petal at four growing stages among different Paeonia lactiflora cultivars were conducted using qPCR technology. The results showed that the expression level of CHI gene exhibited difference in petal tissues from the same cultivar and showed a rise trend from flower bud stage to wither stage; For different cultivars, the expression level of CHI gene in petal tissues of Tongyunjinyan was apparently higher than that of Jinhui and Tongyunjinyan at the same growing stages, furthermore, it was apparently higher in inner petals than that in outer petals for the same cultivars at same growing stages. Considering color difference in inner and outer petals at different growing stages and different Paeonia lactiflora cultivars, it was speculated that the expression level of CHI gene might be related to the color formation of petals in Paeonia lactiflora. The result could provide theoretical guidance for further research on the CHI function and genetic improvement to flower color of Paeonia lactiflora.