Celiac disease (CD) can be triggered in susceptible individuals by the consumption of gluten, a complex storage protein mixture present in wheat, rye and barley. There is no specific reference material (RM) available for barley and this leads to inaccurate quantitation of barley gluten in supposedly gluten-free foods. Therefore, the aim was to select representative barley cultivars to establish a new barley RM. The relative protein composition of the 35 barley cultivars averaged 25% albumins and globulins, 11% d-hordeins, 19% C-hordeins, and 45% B/γ-hordeins. The mean gluten and protein content was 7.2g/100g and 11.2g/100g, respectively. The prolamin/glutelin ratio (1:1) commonly used in ELISAs to calculate the gluten content was found to be inappropriate for barley (1.6±0.6). Eight cultivars suitable as potential RMs were selected to ensure a typical barley protein composition and improve food safety for CD patients.