Boron (B) is an essential mineral element for plant growth, and the seed B pool of crops can be crucial when seedlings need to establish on low-B soils. To date, it is poorly understood how B accumulation in grain crops is genetically controlled. Here, we assessed the genotypic variation of the B concentration in grains of a spring barley (Hordeum vulgare L.) association panel that represents broad genetic diversity. We found a large genetic variation of the grain B concentration and detected in total 23 quantitative trait loci (QTLs) using genome-wide association mapping. HvNIP2;2/HvLsi6, encoding a potential B-transporting membrane protein, mapped closely to a major-effect QTL accounting for the largest proportion of grain B variation. Based on transport studies using heterologous expression systems and gene expression analysis, we demonstrate that HvNIP2;2/HvLsi6 represents a functional B channel and that expression variation in its transcript level associates with root and shoot B concentrations as well as with root dry mass formation under B-deficient conditions.