Eriocaulon buergerianum Koern. is the botanical source for the Chinese herbal medicine Gu-Jing-Cao. Other Eriocaulon species are also used as the same herb in local areas and are difficult to be differentiated. In order to improve the quality control of Gu-Jing-Cao, chemical constituents of E. buergerianum and adulterating species were analyzed by high-performance liquid chromatography with diode array detection and electrospray ionization tandem mass spectrometry (HPLC-DAD-ESI-MS(n)). The 70% methanol extracts were separated on a Zorbax SB-C(18) column and eluted with acetonitrile-water (each containing 0.1% formic acid). The compounds were identified by ion-trap mass spectrometry in both positive and negative ion modes. From E. buergerianum, E. faberi, E. sexangulare and E. cinereum, a total of 72 compounds were characterized, including 37 flavonols, 6 flavones, 4 isoflavones, 6 xanthones, 14 naphthopyranones, 3 phenolic acids, and 2 other flavonoids. Chemical variation of these four species were studied at three tiers, HPLC fingerprinting, quantitation of six major flavonoids.