Although β-glucans in cereals are desirable as healthy diet components, high levels of β-glucans in malting barley are unacceptable because they can cause unsatisfactory degradation of cell walls during malting. The aim of this study was to investigate the β-glucan content in twelve Croatian and two German barley varieties at three representative locations in Eastern Croatia over three consecutive seasons (2012)(2013)(2014). Total β-glucan content in barley samples was determined using enzymatic method. Most of the investigated barley varieties had total β-glucan content lower or significantly lower than 4%. Furthermore, a distinct and clear genotype influence was noticed. No significant difference was found between years, but between locations Osijek and Tovarnik.