This study examined the potential of two winter hulless barley varieties, Eve and VA125, as feedstock for ethanol production. Starch and s-glucan contents of the barley samples were analyzed using official analytical techniques. Both whole-grain and flour samples were hydrolyzed by enzymes and heat treatment preceding fermentation experiments. A simultaneous saccharification and fermentation method was used to convert starch to ethanol. Eve variety had higher starch content than that of VA125. The s-glucan content of VA125 was higher than that of Eve. The highest ethanol conversion efficiency, 88.6%, was attained with the mash prepared from whole-grain Eve. This study demonstrated that winter hulless barley can be a viable feedstock for bio-ethanol production with similar starch content to corn. Dry milling can be used to obtain high starch content flour fractions from barley grain. It is possible to produce ethanol from winter barley varieties with acceptable conversion yields. Optimization of dry milling and ethanol conversion process parameters could improve the economic feasibility of barley-to-ethanol conversion operations.