According to different processing methods and growth conditions, the dry roots of Paeonia lactiflora Pallas are generally classified as Radix Paeoniae Alba (Bai-shao) and Radix Paeoniae Rubra (Chi-shao), which have been officially recorded under different monographs in all editions of Chinese Pharmacopoeia. Although deriving from the same plant origin, the two herbs have individual chemical characteristics, which eventually result in their different efficacies. To better understand their clinical effects, it is important to display the similarity and differentiation of entire chemical patterns of the two drugs in detail. In the present study, the high performance thin layer chromatographic fingerprints combining digital scanning profiling were developed using two suites of mobile phases and derivatization reagents for the 'high polarity component' and 'lipophilic component' fractions contained in the herbal drugs, respectively. The unique properties of the HPTLC fingerprints were validated by analyzing twelve batches of Bai-shao and Chi-shao. Although the HPTLC fingerprint images of the two herbs were similar, significant differences including the presence or absence of characteristic bands and the respective different ratios of the chemical distribution could directly distinguish between Bai-shao and Chi-shao. Furthermore, the corresponding digital scanning profiles can be used as an easy tool for quantifiable comparison among the samples.