Metabolic profiling was carried out in the forage grass Lolium perenne L. (perennial ryegrass) to uncover mechanisms involved in the plants response to water stress. When leaf and root materials from two genotypes, with a contrasting water stress response, were analysed by GC–MS, a clear difference in the metabolic profiles of the leaf tissue under water stress was observed. Differences were principally due to a reduction in fatty acid levels in the more susceptible Cashel genotype and an increase in sugars and compatible solutes in the more tolerant PI 462336 genotype. Sugars with a significant increase included: raffinose, trehalose, glucose, fructose and maltose. Increasing the ability of perennial ryegrass to accumulate these sugars in response to a water deficit may lead to more tolerant varieties. The metabolomics approach was combined with a transcriptomics approach in the water stress tolerant genotype PI 462336, which has identified perennial ryegrass genes regulated under water stress.