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β-glucan content of barley cultivars collected from various areas of China, as well as from Canada and Australia, were assayed by an enzymatic method. Meanwhile a multi-location trial was conducted to determine β-glucan content of eight barley cultivars in each location. For 164 cultivars, originating from China, β-glucan content ranged from 2.98% for Sumei 21 to 8.62% for QB25, with a mean of 4.58%. Tibet barleys, all of the hull-less type, had the highest β-glucan content. Winter barley cultivars from various areas of China had basically the same β-glucan content as those from Canada and Australia. The seeds produced in Hangzhou had lower β-glucan contents than those produced locally. The effects of cultivars, environments (locations) and their interactions on β-glucan content variation was all highly significant, with the cultivars having the largest effect. The highly significant interaction between cultivars and environment suggests that it is important to plant the appropriate cultivar in a particular area in order to obtain barley seeds with reasonable β-glucan contents.