The objective of this study was to evaluate the variability of physiological performances of Castanea sativa Mill. in relation to drought tolerance, among and within European populations coming from contrasting environmental conditions. Forty-eight open-pollinated families from a stratified sample (temperature/precipitation) of six naturalized populations from Spain, Italy and Greece were grown for one growth period under two temperature regimes (25 and 32 °C), in combination with two watering regimes in growth chambers. Complementary to growth traits analysed in a previous study, carbon isotope discrimination (Δ), a complex physiological trait involved in acclimation and adaptive processes, was studied. anova indicated significant Δ variability for C. sativa populations across Europe and, thereby, variation in adaptedness to drought. The European pattern of Δ variability matches the previously reported one for the centre of origin of C. sativa (Ponto-Caucasian region). This suggests that common mechanisms of drought adaptedness, involving both genetic and physiological determinants, give C. sativa the capacity to colonize a wide range of site conditions. The highest Δ values, indicating the lowest water-use efficiency (WUE), were found within each treatment for populations originating from Mediterranean drought-prone sites. These populations also had the highest phenotypic plasticity of Δ. Significant among-family genetic variation in Δ was found. The heritability based on the joint anova was estimated at 0.31 ± 0.07. The estimates of the coefficients for the additive variance varied in the range 2.6–4.0%, suggesting possibilities for selection on WUE and adaptedness to drought. The genetic correlations between Δ and growth traits were generally strong and negative, especially in the two high temperature treatments.