An ultra performance liquid chromatography (UPLC) coupled with quadrupole time-of-flight mass spectrometry (Q-TOF/MS) method has been optimized and established for the rapid analysis of the alkaloids in 22 samples originating from five Uncaria (U.) species. The accurate mass measurement of all the protonated molecules and subsequent fragment ions offers higher quality structural information for the interpretation of fragmentation pathways of the various groups of alkaloids. A total of 19 oxindole alkaloids, 16 indole alkaloids and 1 flavone were identified by co-chromatography of the sample extract with authentic standards, comparison of the retention time, characteristic molecular ions and fragment ions, or were tentatively identified by MS/MS determination. Moreover, the method was validated for the simultaneous quantification of the 24 components within 10.5 min. The potential chemical markers were identified for classification of the U. species samples by principal component analysis (PCA) and orthogonal partial least squared discriminant analysis (OPLS-DA). The results demonstrate the similarity and differences in alkaloids among the five U. species, which is helpful for the standardization and quality control of the medical materials of the U. Ramulus Cum Unics (URCU). Furthermore, with multivariate statistical analysis, the determined markers are more definite and useful for chemotaxonomy of the U. genus.