Soil salinity is a major abiotic stressor and a serious threat to global food security. Biochemical compounds synthesised by plants ameliorate the effects of such stressors in characteristic ways. It is crucial to unravel the key components of the salt tolerance network to engineer salt-tolerant genotypes of crops. For the elucidation of the metabolic contribution to salinity response (long-term stress under 100 mM NaCl) in selected cultivars, we used a gas chromatography mass spectrometry approach. The metabolic profiling of grains yielded sixty-five metabolites. By comparing a salt-sensitive cultivar of barley (Scope) to a salt-tolerant one (GrangeR), we identified six and twenty significant metabolites in salt-stressed seeds of Scope and GrangeR, respectively. These included amino acids, sugars, sugar derivatives and organic acids. Whilst the concentrations of fructose reduced, glycine, glucitol and urea accumulated under salt stress in both varieties. Amino acids such as phenylalanine, tryptophan and tyrosine, sugar acids such as galactaric acid and glucuronic acid, and several other key meta-bolites accumulated in GrangeR only. These metabolites are relevant to metabolic pathways of amino acid metabolism, energy metabolism, carbohydrate metabolism, glycolysis, gluconeogenesis, pyruvate metabolism, shikimate pathway, TCA cycle, inositol phosphate metabolism and galactose metabolism. Hence these com-pounds could be potentially used as biomarkers of abiotic stress responses and effects on grain quality and yield parameters. This work provides greater insights into functional metabolism in response to salinity stress and may help to dissect patterns associated with salt responses. Metabolites identified in this work could serve as accurate markers for salt-tolerant crop selection in breeding programs.