Highland barley (HB) was subjected to dry-, semidry-, wet-milling methods and assessed for flour physico-chemical properties and eating quality of corresponding sugar-free cookies. Results showed that there were significant differences between different milled flours in damaged starch content, particle size, hydration, pasting properties, and color. High a* values and poor hydration/pasting properties of wet-milled flours were associated with its smallest particle size and lowest content of damaged starch (25.3%), beta-glucan (1.87%), and dietary fiber (10.87%), resulting in dark brown color, slightly high spread ratio, low hardness, and fast digestibility of the corresponding cookies. Conversely, the low digestibility of the cookies (predicted glycemic index 58.85) pre-pared from dry-milled flours was attributed to the higher content of dietary fiber, beta-glucan and V-type starch -lipid complex, which would affect enzyme accessibility and may be beneficial for making HB sugar-free cookies. This study is expected to promote the development of HB functional foods.