Polygalaxanthone III, a xanthone glycoside that is a major constituent of “Polygala Root” ( Polygala tenuifolia roots, Onji in the Japanese Pharmacopoeia), has been used as a standard in the quality control of crude drugs. However, we previously noted differences in the chromatographic properties of one of three samples of polygalaxanthone III. Therefore, standardization of the standard itself is extremely important. The structures of three standard samples commercially available as polygalaxanthone III were characterized by LC/MS and NMR. LC/MS analysis revealed that two molecular types exist. Both types are chromatographically separable but have an identical mass number with distinguishable MS/MS spectra. One dimensional (1D)-NMR analyses demonstrated that both had the same xanthone moiety and heteronuclear multiple bond correlation (HMBC) analyses revealed that they are structural isomers at the connecting position of glucose to apiose 1-position. Consequently, the isomers were identified as polygalaxanthone III and its regioisomer, polygalaxanthone XI. Based on the findings, we recommend using the LC-MS/MS detection method, which discriminates polygalaxanthone III and XI, to confirm the quality of the standard.