The process of breeding superior barley (Hordeum vulgare) varieties for the brewing industry is lengthy and expensive. The ability to identify new metabolite markers for grain quality would substantially expedite selecting superior varieties for the brewing. We developed a high-throughput metabolite screen to evaluate 72 varieties (36 2-row type and 36 6-row type) across two growing locations. Approximately 4,800 metabolites were detected in each sample using ultra performance liquid chromatography coupled to mass spectrometry, and variation by barley genotype, row-type, growing location, and genotype by location interactions was observed. Metabolite data was integrated with approximately 2,000 SNP markers and 18 quality traits to identify small molecules and gene regions associated with grain quality, as well as metabolites affected by growing environment. This approach shows how metabolite markers can be used in conjunction with genetic markers to provide the opportunity for a high-throughput screen of hundreds of varieties and growing environments, which can significantly advance breeding barley with enhanced brewing characteristics.