The use of genome-wide association studies (GWAS) to detect quantitative trait loci (QTL) controlling complex traits has become a popular approach for studying key traits in crop plants. The goal of this study was to identify the genomic regions of barley (Hordeum vulgare L.) that impact five agronomic and one quality trait in U.S. elite barley breeding lines, as well as to identify markers tightly linked with these loci for further use in barley improvement. Advanced recombinant inbred lines submitted to the U.S. Barley Coordinated Agricultural Project (CAP) were genotyped using a platform of 3072 single nucleotide polymorphism (SNP) markers from the barley oligonucleotide pool assays (BOPAs) 1 and 2. In each of 4 yr, approximately 770 lines were evaluated in a replicated, randomized complete block design under both irrigated and dryland conditions. This gave an overall population size of > 3000 lines, which we analyzed in a hierarchical fashion, including analyzing the lines in aggregate using a mixed model to account for population structure and relatedness among the lines. We identified 41 significant marker–trait associations, of which 31 had been previously reported as QTL using biparental mapping techniques; 10 novel marker-trait associations were identified. The results of this work show that genes with major effects are still segregating in U.S. barley germplasm and demonstrate the utility of GWAS in barley breeding populations.