Rattail fescue (Vulpia myuros (L.) C.C. Gmel.) is a self-pollinating winter annual grassy weed of winter annual crops. The problems with V. myuros 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 V. myuros control has been reported following glyphosate applications. Experiments were performed to study the effectiveness of glyphosate on V. myuros, and determine the causes of the lower performance of glyphosate on V. myuros compared to other grass weeds. Estimated GR50 values demonstrated that V. myuros was less susceptible to glyphosate than Apera spica-venti 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 V. myuros was not caused by lower retention as previously suggested. A significantly lower shikimic acid accumulation in V. myuros compared to A. spica-venti was associated with a higher activity of the EPSPS enzyme in V. myuros. Nevertheless, the relative responses in EPSPS activity to different glyphosate concentrations were similar in the two grass species, which indicate that EPSPS from V. myuros is as susceptible to glyphosate as EPSPS from A. spica-venti suggesting no alternation in the binding site of EPSPS. The results from the current study indicate that V. myuros is less susceptible to glyphosate compared to A. spica-venti, and the low susceptibility of V. myuros is caused by an increased EPSPS enzyme activity.