The depth of water extraction is a key trait in grassland species. For comparing individual plant forms in the field, different ecological studies have proven the efficiency of the isotopic signal of water (δ18O). To date, there has been no assessment of the aforementioned method within closely related grasslands species. For this study, Festuca arundinacea and Lolium multiflorum plants were grown in nutrient solution and the δ18O of different plant compartments were compared to the δ18O of the solution under both high and low transpiration conditions. In both species, the basal part of the tillers, excluding the eldest sheath, closely reflected the isotopic water composition of the nutrient solution, whereas other parts of the plant exhibited a significant enrichment in 18O as a consequence of transpiration. In a second experiment, using the genetic variability within plants obtained from crossing F. arundinacea with Lolium multiflorum, the study explored the variability of the depth of water extraction and δ18O under conditions of soil water deficit. A plant nursery of hybrids was established in a greenhouse with plants sewn directly in the soil. Ten neutron probe access tubes were placed close to ten individual plants, which were sampled twice at 38 day intervals, following a period of 14 and 52 days of water deprivation. On these same dates, soil samples were extracted down to 1 meter in the vicinity of the 10 studied plants. Water was fully extracted from both plant and soil samples and the isotopic composition was measured. The predawn leaf water potential exhibited large variations in water status among the ten plants studied. The measured plant δ18O were similar to the values computed from estimated soil water extraction profiles (using neutron probe measurements) and soil δ18O of the soil water profile. On the second date of sampling, the δ18O and leaf water potentials of three individual plants (L. multiflorum) were observed throughout the day, revealing a gradual decrease in 18O content as the plant water potential decreased. The role of short term changes in the soil–root hydraulic conductance on depth of water extraction is discussed in this study.