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β-glucan is an important trait to be improved in barley breeding programs, as it greatly affects the quality of the end products when barley grains are used as raw material of feed or malt production or consumed as food for human. Although the genes associated with β-glucan synthesis have been identified, genetic regulation of β-glucan accumulation in barley grains is still completely unclear. In this study, 100 accessions from International Barley Core Selected Collection (BCS) were planted in two environments for two consecutive years to determine the genotypic variation of grain β-glucan content. A genome-wide association study (GWAS) identified 14 stable marker-trait associations (MTAs) (−Log10(P)> 4) for grain β-glucan content. Significantly positive correlation was found between grain β-glucan content and the number of favorable alleles of 14 stable MTAs. Seven putative candidate genes encoding some enzymes in glucose metabolism were found to be associated with β-glucan content. One of the putative genes, HORVU6Hr1G088380, could be an important gene controlling barely β-glucan content, with the SNPs being closely linked in all tested accessions and divided into two haplotypes. High resolution melting (HRM) analysis of the first SNP suggested that the HRM-SNP marker is valid for marker-assisted selection in barley breeding. This study provides useful information for the genes and markers related to grain β-glucan content in barley. [ABSTRACT FROM AUTHOR]