In wheat, β-glucan (BG) content represents an important component of dietary fibre with important health benefit. Among cereals, barley, oats and rye have the highest BG content in the grain (from 3 to 10%), while bread and durum wheat contain percentages lower than 1%. Screening genetic resources for improving wheat BG can contribute to human health. In this study, a complete series of wheat–barley addition lines were evaluated for the BG content in replicated field trials for 3 years to determine how the added chromosome influence the BG in wheat. On the overall, the BG varied among the addition lines, and the effect of individual barley chromosomes on the wheat background was evident in each experiment. Significant higher BG than Chinese Spring was found in four addition lines (CS-1H/1HS-6H, CS-2H, CS-6H and CS-7H), while three lines (CS-3H, CS-4H and CS-5H) showed lower amount than the wheat parental line. Particularly, CS-7H had significant high BG in all the years considered, with percentage increments of the mean values ranging from 17.2 to 48.1% in comparison to Chinese Spring. Though BG in wheat grain increased in some addition lines, it was still much lower than in barley. Results are discussed in relation to the genes controlling the synthesis and breakdown of BG in the wheat background.