The use of glyphosate-tolerant perennial ryegrass (Lolium perenne L.) (PRG) cultivars JS501 and Replay provides turfgrass managers a unique option for annual bluegrass (Poa annua L.) (ABG) control. Both cultivars can tolerate a maximum glyphosate rate of 0.81 kg·ha−1 acid equivalent (a.e.) after establishment under optimal growing temperatures (16 to 24 °C). However, tolerance to applications made immediately after germination and during low air temperatures has received limited investigation. Therefore, objectives of this research were to determine the seedling tolerance and low-temperature response after a fall season glyphosate application to both cultivars. Field trials were conducted in Idaho and Oregon. For the fall application response trial in Idaho, glyphosate was applied at 0, 0.15, 0.29, 0.58, 1.16, 1.74, 2.32, and 3.48 kg·ha−1 a.e. In Oregon, glyphosate was applied at 0, 0.15, 0.29, 0.44, 0.58, 1.16, and 3.48 kg·ha−1 a.e. At both sites, applications were made between late September and early October. To determine seedling tolerance, both cultivars were sprayed with glyphosate at the one-leaf stage (LS), two LS, three LS, or four LS at rates of 0, 0.15, 0.29, and 0.58 kg·ha−1 a.e. Across all trials, ratings included PRG color, cover, and injury. At both trial locations, regression analysis revealed a rate of ≈0.27 kg·ha−1 a.e. was required to cause 20% leaf firing in the fall application response trial. In the seedling tolerance trial, glyphosate applied at 0.58 kg·ha−1 a.e. at the one LS, two LS, and three LS had color ratings 8.0 or greater; however, color ratings dropped to 4.6 when an application was made at the four LS. Based on the environmental conditions of each trial, results suggest glyphosate applications greater than 0.27 kg·ha−1 a.e. as minimum air temperatures approach 0 °C should be avoided. Also, applications should be avoided at the three to four LS if the application rate is greater than 0.29 kg·ha−1 a.e.