To explore rapidly the potential chemical markers for differentiating Radix Paeoniae Alba and Radix Paeoniae Rubra, a method is proposed based on ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS) coupled with multivariate statistical analysis. Batches of commercial samples were analyzed by UPLC-Q-TOF/MS. The datasets of t(R)-m/z pair, ion intensities and sample codes were further processed with orthogonal partial least squared discriminant analysis (OPLS-DA) to compare holistically the difference between these two kinds of samples. Then statistics were used to generate an S-plot, in which the variables (t(R)-m/z pair) contributing most to the difference were clearly depicted as points at the two ends of "S", and the components correlated to these ions should be regarded as the chemical markers. The identities of the most changed markers can be identified by comparing the mass/UV spectra and retention times with those of reference compounds and/or tentatively assigned by matching empirical molecular formulae with those of known compounds published in the literature. Using this proposed approach, albflorin, paeoniflorin, oxypaeoniflorin, benzoylpaeoniflorin, galloylalbiflorin and paeoniflorigenone were found to be the differentiating components for discrimination of Radix Paeoniae Alba and Radix Paeoniae Rubra. Moreover, paeoniflorin sulfonate and its isomer, isomaltopaeoniflorin sulfonate, were found to be the characteristic markers for all Radix Paeoniae Alba samples that were processed by sulfurdioxide gas fumigation. The results suggested that this newly established approach could be used to explore rapidly the potential chemical markers for herbs with similar chemical characteristics.