Pre-harvest glyphosate is applied to cereal grains to remove weeds. However, it has been claimed that oat compositions are affected by pre-harvest glyphosate. Research was conducted to evaluate differences in properties of β-glucan in the treated versus untreated oat groats. Two oat cultivars (Rockford and Souris) were grown at Minot and Prosper, North Dakota in 2015, and glyphosate was sprayed during the soft dough stage, physiological maturity stage, or not applied. β-Glucan viscosity was not significantly (p > 0:05) affected by treatment at soft dough or physiological maturity stages. Use of glyphosate at the soft dough stage significantly (p < 0:05) reduced the percentages of β-glucan content and solubility versus untreated samples. Treatment at soft dough and physiological maturity stages significantly (p < 0:05) increased β-glucan molecular weights compared to untreated controls. Therefore, glyphosate can be applied at the physiologically mature stage of grain development because β glucan properties from the groats were not negatively affected.