The length of the light period before harvest and air temperature are known to affect forage nutritive value, particularly the concentration of non-structural carbohydrates (NSC). Our objective was to assess the effect of the length of the light period before harvest on yield, digestibility, and concentrations of N, acid detergent fiber (ADF), neutral detergent fiber (NDF), and NSC of timothy (Phleum pratense L.) grown under different temperature regimes. Growth temperatures were chosen to represent existing conditions in contrasted areas of eastern Canada, and predicted climate change over the next 50 years. Timothy was grown in 15-cm diameter pots in three growth chambers adjusted to day/night temperatures of 17/5, 22/10, or 28/15°C under a 15-h photoperiod. Timothy was either harvested in the morning (AM, 8h00) or the afternoon (PM, 15h00). The same experiment was repeated a second time using the same three growth chambers. No significant interaction occurred between air temperature and AM-PM harvesting. The PM-harvested timothy had significantly higher NSC (41.3 vs. 32.0 g/kg DM) and sucrose (28.5 vs. 19.4 g/kg DM) concentrations, and lower ADF (325 vs. 330 g/kg DM) and NDF (530 vs. 540 g/kg DM) concentrations than AM-harvested timothy. Digestibility of DM and NDF, and N concentration of AM and PM-harvested timothy did not differ. With increasing day/night temperature from 17/5°C to 28/15°C, timothy DM yield decreased from 7.9 to 4.7 g/pot, DM digestibility decreased by 51 g/kg DM, and NDF digestibility decreased by 96 g/kg NDF but lignin, ADF, and NDF concentrations were not affected. The NSC concentration at 17/5 and 22/10°C did not differ but it increased at 28/15°C, presumably because of a heat stress. The nutritive value of timothy is therefore greater when harvested in PM and grown under cool temperatures (17/5°C).