Two-row and six-row barley (Hordeum vulgare L.) cultivars, originating from eastern and western Canada were grown in six environments across Canada. The grain was studied for the effects of environment and cultivar on the total mixed-linkage β-glucan content (TBG, g hg−1 DM). Environment significantly affected TBG content in barley. TBG content differed among the 32 eastern or 43 western Canadian cultivars but the mean TBG for the eastern cultivars was not different from the mean of western cultivars. All eastern cultivars were hulled-feed types, and their TBG ranged from 3.31 in Micmac to 4.09 in Birka for two-row barleys, and from 3.78 in OAC Kippen to 4.41 in Maskot for six-row barleys. Western hulled cultivars which included feed and malt types, contained TBG ranging from 3.37 in Manley to 4.14 in Betzes for two-row barleys, and from 3.71 in Heartland to 4.42 in Tankard for six-row barleys. The two-row hulless cultivar, Condor, contained more TBG (4.69) than the other two hulless cultivars, CDC Richar...