Background: Chinese medicinal herbs may use more than one species of Curcumae Radix (Yujin) is the tuberous roots of Curcumae wenyujin, C. kwangsiensis, C. phaeocaulis and C. longa. This study aimed to characterize the chemical profiles of these different species of Curcumae Radix, and develop a method for rapid discrimination of these species by ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC/Q-TOFMS) combined with multivariate statistical analysis. Methods: The metabolomes of 33 different batches of Curcumae Radix derived from four Curcumae species were profiled by UHPLC/Q-TOFMS. The resulting sample codes, t-m/z pairs and ion intensities were processed by unsupervised principal component analysis (PCA) and supervised orthogonal partial least squared discriminant analysis (OPLS-DA) to characterize the chemical composition of Curcumae Radix across the four different species. Results: Obvious differences were observed in the chemical compositions of the Curcumae Radix samples derived from the four different species according to PCA and OPLS-DA. These results suggested that curcumin, curcumenone, curcumenol and zederone could be used as unique chemical markers for C. longa, C. wenyujin, C. phaeocaulis and C. kwangsiensis, respectively. Conclusions: This study developed a UHPLC/Q-TOFMS method coupled with multivariate statistical analysis to discriminate between Curcumae Radix samples from four different Curcumae species, i.e., C. longa, C. wenyujin, C. phaeocaulis and C. kwangsiensis. Notably, this new approach resulted in the identification of curcumin (a), curcumenone (b), curcumenol (c) and zederone (d) as unique chemical markers for the identification.