Osmoprotectants have been reported to reduce the detrimental effects of various environmental stresses in many different plant species. However, there is little research available concerning pasture species. Two experiments were undertaken with the aim of quantifying the effect of surface applications of exogenous glycine betaine (GB) on herbage production and quality of perennial ryegrass (Lolium perenne L.)–white clover (Trifolium repens L.) pastures during periods of moisture stress and cold temperatures over 2 years. Pastures fertilised with GB were compared with unfertilised pastures and pastures fertilised with nitrogen (N). Rates of 0.5, 1.0 and 1.5 kg GB/ha.defoliation were applied in experiment 1 and 5 kg GB/ha.defoliation was applied in experiment 2. Surface applications of GB did not significantly affect herbage production relative to unfertilised pastures; herbage yields averaged 12 248 and 12 693 kg DM/ha over 11 months in experiment 1, and 7253 and 7177 kg DM/ha over 6 months during summer and autumn in experiment 2, for the unfertilised control and GB, respectively. During both experiments, herbage quality parameters were not affected by GB application, although the proportion of white clover in the sward between summer and winter during experiment 1 was greater (P < 0.01) in plots treated with GB than in untreated plots. Application of N fertiliser increased (P < 0.001) herbage production, but did not consistently affect herbage quality. The failure of surface applications of exogenous GB to improve the herbage production or quality of perennial ryegrass–white clover pastures suggests that it is not an appropriate method to enhance plant tolerance to environmental stress at the concentrations applied in these studies.