Global change is bringing barley with more frequency of suffering from high temperature. However, little has been known about the influence of high temperature on malt quality traits. In this study, we investigated the impact of 1-wk heat stress (32°C/26°C, day/night, 12 h/12 h) initiating from the 7th (HT7) and 14th (HT14) days after heading on some grain and malt quality traits of two barley cultivars. In comparison with normal temperature (24°C/18°C, day/night, 12 h/12 h), heat stress significantly reduced kel weight, seed setting rate and grains per spike: HT7 having a larger effect than HT14.Meanwhile, total protein and β-glucan contents, and β-amylase and limit dextrinase activities were significantly increased under high temperature, with HT7-treated plants showing larger changes. Moreover, the different changes of four protein fractions under heat stress were found in the two barley cultivars, indicating the possibility of breaking positive association between protein content and enzyme activity.