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Summary: Genetic biofortification requires knowledge on natural variation and the underlying mechanisms of micronutrient accumulation. We therefore studied diversity in grain micronutrient concentrations and spatial distribution in barley (Hordeum vulgare), a genetically tractable model cereal and an important crop with widespread cultivation.We assembled a diverse collection of barley cultivars and landraces and analysed grain micronutrient profiles in genebank material and after three independent cultivations. Lines with contrasting grain zinc (Zn) accumulation were selected for in‐depth analysis of micronutrient distribution within the grain by micro‐proton‐induced X‐ray emission (μ‐PIXE). Also, we addressed association with grain cadmium (Cd) accumulation.The analysis of > 120 lines revealed substantial variation, especially in grain Zn concentrations. A large fraction of this variation is due to genetic differences. Grain dissection and μ‐PIXE analysis of contrasting lines showed that differences in grain Zn accumulation apply to all parts of the grain including the endosperm. Cd concentrations exceeded the Codex Alimentarius threshold in most of the representative barley lines after cultivation in a Cd‐contaminated agricultural soil.Two important conclusions for biofortification are: first, high‐Zn grains contain more Zn also in the consumed parts of the grain; and second, higher micronutrient concentrations are strongly associated with higher Cd accumulation. [ABSTRACT FROM AUTHOR]