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Rattail fescue (<i>Vulpia myuros</i> (L.) C.C. Gmel.) is a self-pollinating winter annual grassy weed of winter annual crops. The problems with <i>V. myuros</i> are mostly associated with no-till cropping systems where glyphosate application before sowing or emergence of the crop is the most important control measure. Ineffective <i>V. myuros</i> control has been reported following glyphosate applications. Experiments were performed to study the effectiveness of glyphosate on <i>V. myuros,</i> and determine the causes of the lower performance of glyphosate on <i>V. myuros</i> compared to other grass weeds. Estimated GR₅₀ values demonstrated that <i>V. myuros</i> was less susceptible to glyphosate than <i>Apera spica-venti</i> regardless of the growth stage. Within each species, glyphosate efficacy at different growth stages was closely related to spray retention. However, the low susceptibility to glyphosate in <i>V. myuros</i> was not caused by lower retention as previously suggested. A significantly lower shikimic acid accumulation in <i>V. myuros</i> compared to <i>A. spica-venti</i> was associated with a higher activity of the EPSPS enzyme in <i>V. myuros</i>. Nevertheless, the relative responses in EPSPS activity to different glyphosate concentrations were similar in the two grass species, which indicate that EPSPS from <i>V. myuros</i> is as susceptible to glyphosate as EPSPS from <i>A. spica-venti</i> suggesting no alternation in the binding site of EPSPS. The results from the current study indicate that <i>V. myuros</i> is less susceptible to glyphosate compared to <i>A. spica-venti,</i> and the low susceptibility of <i>V. myuros</i> is caused by an increased EPSPS enzyme activity.