Recent research has shown that plant acclimation to diverse patterns of light intensity modifies the dynamics of their stomatal response. Therefore, whether plants are grown in controlled conditions or in the field may impact their stomatal dynamics. We analysed the stomatal dynamics of two Populus euramericana and two Populus nigra genotypes grown in the field under contrasting water availability. By comparing their stomatal dynamics with that of the same genotypes grown in a glasshouse, we were able to test whether differences between these growing conditions interacted with genotypic differences in affecting stomatal dynamics and responses to soil water deficit. We found that, despite higher stomatal density and smaller size, in the field stomatal dynamics were much slower than in the glasshouse. Overall, differences among genotypes and their response to soil water deficit were much less pronounced in the field compared with the glasshouse. These results indicate that stomatal dynamics are regulated by both genotype-specific and environmental factors. Moreover, having slower stomata may be advantageous under some conditions. While stomatal dynamics were linked with whole-plant transpiration per leaf area in both experiments, the contribution of stomatal morphology varies dependent on the environmental conditions.