Quality traits of barley (Hordeum vulgare L.) grain, such as starch and protein contents, are important factors that determine grain end-use value. In barley breeding, improved quality traits have long been a critical goal. The objective of this study was to identify underlying genetic locations for grain quality traits and mine the related candidate genes. In this study, 250 barley accessions were evaluated for key grain quality traits, including protein content (GPC), starch content (GSC) and fibre content (GFC) across a two-year period. Through genome-wide association study (GWAS) analysis, we identified 145 significant SNPs associated with grain quality traits. Additionally, two materials showed significant differences in grain quality were further selected for transcriptome analysis. Through a comparison analysis of barley grain, 4636 and 3684 differentially expressed genes (DEGs) were identified at 21 days post anthesis (DPA) and 28 DPA, respectively. These DEGs mainly participate in carbon fixation in photosynthetic organisms, starch and sucrose metabolism, and so on. By integrating GWAS and RNA-Seq data analysis approaches, we identified 17 candidate genes primarily involved in hydrolysis of starch, glycosyltransferase and post-translational modification. Altogether, 27 stable loci significantly associated with grain quality traits were detected, and 17 candidate genes related to grain quality were screened. These findings provide valuable insights for gene cloning related to grain quality and server as a reference for high-quality barley cultivars.