To evaluate the relationship between tree peony cultivars and the quality consistency of Cortex Moutan, a sensitive, reliable, and validated method based on high-performance liquid chromatography-electrospray ionization/mass spectrometry was developed for the quantitative analysis of paeonol and chemical fingerprinting of Cortex Moutan. Results from quantitative analysis showed that the content of paeonol in Paeonia ostii “Feng Dan” was the highest (24.51 ± 0.83 mg/g), followed by Paeonia suffruticosa “Luoyang Hong” (14.29 ± 0.76 mg/g), P. suffruticosa “Taiping Hong” (13.99±1.13 mg/g), and P. suffruticosa “Zhaofen” (13.08±0.85 mg/g). Paeonia ostii “Luoyang Feng Dan” was found to have the lowest content (8.76±0.46 mg/g) of paeonol. In qualitative analysis, 5 tree peony cultivars collected from different plantations in China were used to establish the fingerprint. For the fingerprint analysis, 17 characteristic peaks were used to evaluate similarities among tree peony cultivars, and they were found to show similarities. In short, the results of quantitative and qualitative analyses suggested that there was no significant difference in the chemical composition of Cortex Moutan from different tree peony varieties; however, there were significant differences in the levels of chemical components. The method developed in this study provides an important reference to establish a quality control method for other related traditional Chinese medicinal preparations.