Climatic events predicted for this century should involve drier and warmer summers, such as those that occurred in 2003 and 2005 in France. The higher temperatures could become a limiting factor for the regeneration of Populus nigra L. species. During establishment and development in summer period, seedlings must be able to resist high temperatures (until 57.8°C on sand on a Loire island in July 2009). Therefore, maintaining a high transpiration rate (E) for leaf cooling (and therefore a high stomatal conductance to water vapour (gs)), may be a prerequisite for seedlings to survive under high temperatures. To test this hypothesis, we used bulk leaf carbon isotope discrimination (Δ) as a time-integrated index of gas exchange activity (Farquhar et al. 1989). The objectives of this study were (1) to explore the genetic variability of ∆ among P. nigra seedlings from different female trees coming from contrasting sites and (2) the plasticity in response to two contrasting temperatures. Seeds from 16 open-pollinated females originating from the Loire river (France) and from the Paglia river (Italy) were first grown during 7 weeks in two growth chambers at 25°C. At this time (t0), one chamber remained at 25°C and in the second one, the temperature progressively increased until 43°C until the first symptoms of wilting appeared (t1). At t0, an important genetic variability for ∆ (∆t0 25°C) was measured ranged from 24.0‰ to 28.0‰ in both chambers, but no provenances differences were detected. At t1, an important genetic variability for ∆t1 25°C and ∆t1 43°C was observed ranged from 22.0‰ to 26.0‰ in each chamber and a significant provenance effect was detected. At t1, ∆t1 43°C values were significantly higher (0.5‰) than ∆t1 25°C. Moreover, a significant correlation was detected between the ∆t1 43°C values and the symptoms of wilting seedlings, seedlings totally healthy exhibiting higher ∆t1 43°C values. We conclude, under the hypothesis that ∆ is mainly controlled by gs, as already reported in poplar species (Monclus et al. 2006), maintaining high E and therefore high ∆ may enable seedlings to survive under high temperature.