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Background: Radix Paeoniae Alba, Radix Paeoniae Rubra, and Cortex Moutan are important Chinese herbs. Their bioactivities and efficacies are similar. However, they have different superior benefits clinically; so, a comprehensive investigation of the chemical difference is necessary and is of great importance for more reasonable quality assessment and proper clinical application of these three herbal medicines. Objective: To establish a high-performance liquid chromatography (HPLC) fingerprint method for the quality control of Radix Paeonia Alba, Radix Paeonia Rubra, and Cortex Moutan, and to compare their main constituents. Materials and Methods: The separations of Radix Paeoniae Alba, Radix Paeoniae Rubra, and Cortex Moutan was carried out, respectively, through a gradient elution using a monolithic column and a mobile phase consisting of water (containing 0.1% formic acid) and acetonitrile (containing 0.1% formic acid) at a flow rate of 5.0 ml/min. The detection wavelength was set at 230 nm. The data calculation was performed with CHROMAP v1.51 and Statistical Package for the Social Sciences (SPSS) 18.0 software for principal component analysis. Results: A rapid separation method based on high-performance liquid chromatography with diode-array detection (HPLC-DAD) with monolithic columns and a fingerprint analysis method was established. Fifteen Radix Paeonia Alba, 45 Radix Paeonia Rubra, and 21 Cortex Moutan samples were analyzed and 11 chromatographic peaks were identified. Differences of chromatographic peaks among these three herbal medicines in chemical compositions were revealed. Conclusion: The separation and analysis method are fast and simple, which can be used for chemical fingerprint comparison of Radix Paeonia Alba, Radix Paeonia Rubra, and Cortex Moutan. The results for the evaluation of the three medicines could provide experimental evidence for chemical affinity. [ABSTRACT FROM AUTHOR]