Boron (B) is an essential micronutrient that affects plant growth at either deficient or toxic concentrations in soil. The aim of this work was to investigate the adaptation of barley (Hordeum vulgare) plants to toxic B levels and to increase our understanding of B toxicity tolerance mechanisms. We used a metabolomics approach to compare metabolite profiles in root and leaf tissues of an intolerant, commercial cultivar (cv Clipper) and a B-tolerant Algerian landrace (cv Sahara). After exposure to elevated B (200 and 1,000 microM), the number and amplitude of metabolite changes in roots was greater in Clipper than in Sahara. In contrast, leaf metabolites of both cultivars only responded following 1,000 microM treatment, at which B toxicity symptoms (necrosis) were visible. In addition, metabolite levels were dramatically altered in the tips of leaves of the sensitive cultivar Clipper after growth in 1,000 microM B compared to those of Sahara. This correlates with a gradual accumulation of B from leaf base to tip in B-intolerant cultivars. Overall, there were always greater differences between tissue types (roots and leaves) than between the two cultivars. This work has provided insights into metabolic differences of two genetically distinct barley cultivars and information about how they respond metabolically to increasing B levels.