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L. ruthenicum, which grows mainly in the salinized desert of the Qinghai-Tibet plateau, contains abundant anthocyanins. To date, information on its anthocyanin composition is still limited. In this work, the systematic characterization of anthocyanins in L. ruthenicum was carried out by HPLC-DAD/QTOF-MS/MS. Six mono-acylated anthocyanins isolated from L. ruthenicum were analyzed by ESI-QTOF-MS/MS, and their fragmentation behaviors were investigated and summarized. Different anthocyanins could be identified through the characteristic product ions and their relative intensities. In addition, various rules were applied to cope with the difficulty of rapidly differentiating cis-trans anthocyanin isomers. A spectral rule was first proposed from the UV/Vis spectra of two pairs of cis-trans anthocyanin isomers. This rule provided important information for the identification of the isomers. Based on these rules, sixteen anthocyanins in L. ruthenicum were tentatively characterized, and seven of them were reported in this plant for the first time. In order to validate the method, an anthocyanin was isolated from L. ruthenicum and identified by NMR and MS.