Background: Tibetan wild barley (Hordeum spontaneum L.) has been confirmed to contain elite accessions in tolerance to abiotic stresses, including salinity. However, molecular mechanisms underlying genotypic difference of salt tolerance in wild barley are unknown. Results: In this study, two Tibetan wild barley accessions (XZ26 and XZ169), differing greatly in salt tolerance, were used to determine changes of ionomic, metabolomic and proteomic profiles in the shoots exposed to salt stress at seedling stage. Compared with XZ169, XZ26 showed better shoot growth and less Na accumulation after 7 days treatments. Salt stress caused significant reduction in concentrations of sucrose and metabolites involved in glycolysis pathway in XZ169, and elevated level of tricarboxylic acid (TCA) cycle, as reflected by up-accumulation of citric acid, aconitic acid and succinic acid, especially under high salinity, but not in XZ26. Correspondingly, proteomic analysis further proved the findings from the metabolomic study. Conclusion: XZ26 maintained a lower Na concentration in the shoots and developed superior shoot adaptive strategies to salt stress. The current result provides possible utilization of Tibetan wild barley in developing barley cultivars for salt tolerance. [ABSTRACT FROM AUTHOR]