The production of barley cultivars with malting and brewing quality characteristics is subject to strict grading and technical standards for the end-use market. Environmental and management factors can significantly alter grain quality, and the qualities required for malting. Crop and kernel uniformity are critical factors where variability can exceed the tolerance for meeting malt quality. A practice to address variations in crop maturity is the application of pre-harvest glyphosate. Pre-harvest glyphosate applications can, however, alter malting characteristics in barley, and, if mis-timed, can also reduce yield. A 4-year study at five locations in Alberta and Saskatchewan from 2013 to 2017 was conducted to determine the effects of pre-harvest glyphosate applications on malting barley characteristics. Glyphosate was applied at 900 and 1125 g ae ha⁻¹ on malting barley cultivars ‘CDC Meredith’ and ‘AC Metcalfe’ malting barley at soft dough, hard dough, and physiological maturity growth stages. Yield reductions of up to 12% were observed from glyphosate applications at soft dough, and yield was maximized with applications at physiological maturity. Glyphosate application, at two rates, reduced percentage plump kernels, but did not affect kernel weight or protein concentration. The growth stage of barley plants did not provide an accurate indicator of seed moisture levels at the time of application, which motivated our conclusion that glyphosate applications can be mistimed by inaccurate indicators. The results motivate our questioning of the utility of pre-harvest glyphosate applications, given the adverse effects to barley yield and quality observed, even when applied according to the label instructions.