In order to study observed differences in water status between Trifolium repens L. cv. Regal NC‐R and NC‐S biotypes, tolerant and sensitive to ozone, respectively, and trying to identify possible environmental strains for this widely used ozone biomonitoring system, the response of these two biotypes to a short‐term drought was evaluated. The evolution of both soil and plant water status along with leaf gas exchange parameters, leaf ascorbate peroxidase (EC 1.11.1.11), glutathione reductase (EC 1.8.1.7), superoxide dismutase (EC 1.15.1.11) enzymatic activities and leaf content in total soluble protein, total soluble sugars, proline for an equal number of clones of each biotype under drought and control treatments was registered for 5 days under greenhouse conditions and summer direct sunlight. The results obtained show that the water requirement of sensitive NC‐S clones is higher, as these tend to display higher values of leaf conductance and water use under optimal water availability. What is more, when the short‐term drought is imposed, symptoms of oxidative stress and a negative impact on most of the measured parameters are displayed at an earlier stage in this biotype, proving it to be more sensitive to low water availability. It is therefore suggested that these differences might be linked with the existing variability in ozone sensitivity between the biotypes and that at the same time, they could result in a poorer performance of this biomonitoring system in field assays under conditions of high evapotranspiratory demand, such as those registered during summer time in Southern Europe.