Climate change, as summarised by global warming, poses a substantial challenge to malting barley (Hordeum vulgare L.) production and the malting industries. This is because, in sensitive varieties, heat stress causes grain protein content (GPC) to exceed the maximum acceptable value for malting purposes. One way to avoid the problem is to use cultivars with inherently low-GPC and hence reduce the risk of approaching a GPC above 12 %. The present study aimed to characterise the genetic locus on 5HS linked to GPC in two-rowed barley and to combine it with the locus on 6H identified in six-rowed barley. Saturation mapping with EST-derived SSR markers identified four new markers (UMB704, K03390, UMB707, and HvSMEh297) within the target QTL region, which provides functional markers for selection. Over a 3-year period, doubled haploid lines differing in the presence/absence of the marker alleles at the 5HS locus were characterised for physiological differences related to GPC accumulation. Both genotype groups flowered at about the same time, but during grain filling, the low-GPC group accumulated less nitrogen in the spikes, and more dry matter, than the high-GPC group. The 5HS and 6H QTLs were combined into two-row backgrounds, and preliminary field evaluation showed that almost all of the recombinants had GPC levels that were lower than the commercial check (Schooner) by up to 4 % units. Of the 155 F2-derived F5 lines, 20 had grain protein levels that were lower than that of the low-GPC check (VB0229) by 1–2 %. This represents a new germplasm pool that will be useful for variety development, not just to lower GPC, but also to stabilise GPC variability across different environments.