Background Water use efficiency (WUE) is an important adaptive trait for soil water deficit. The molecular and physiological bases of WUE regulation in crops have been studied in detail in the context of plant breeding. Knowledge for most forest tree species lags behind, despite the need to identify populations or genotypes able to cope with the longer, more intense drought periods likely to result from climate warming. Results We aimed to bridge this gap in knowledge for sessile oak (Quercus Petraeae Matt. L.), one of the most ecologically and economically important tree species in Europe, using a factorial design including two genotypes (low and high WUE) and two watering regimes (control and drought). By monitoring the ecophysiological response, we were able to identify groups of genotypes with high and low WUE. We then performed RNA-seq to quantify gene expression for the most extreme genotypes exposed to two watering regimes. By analyzing the interaction term, we were able to capture the molecular strategy of each group of plants for coping with drought. Regardless of water availability, the high WUE genotypes overexpressed genes associated with drought responses, and the control of stomatal density and distribution, and displayed a downregulation of genes associated with early stomatal closure and high transpiration rate. High-WUE genotypes, thus, coped with drought by fine-tuning the expression of genes with known functions in the regulation of stomatal size, density, movement or aperture and transpiration rate. Conclusion Fine physiological screening of sessile oaks with contrasting WUE, and their molecular characterization i) highlighted subtle differences in transcription between low and high WUE genotypes, identifying key molecular players in the genetic control of this trait, and ii) revealed the genes underlying the molecular strategy that had evolved in each group to cope with water deficit, providing new insight into the value of WUE for adaptation to drought.