Poplar (Populus), the model system in tree research, is a fast-growing and high biomass plant which is promising for energy, paper and pulp production, and for growth in soils contaminated with metals. Contamination of soils and water with heavy metals has become a widespread problem; environmental pollution by excess zinc (Zn), one of the more important contaminants, occurs frequently and yet the responses of Populus to high Zn concentrations are still not clearly understood. We investigated the effects of Zn on the functional and structural parameters in the Populus×euramericana clone I-214 by Zn localization in frozen-hydrated leaves and roots by cryo-scanning electron microscopy (cryo-SEM)/energy-dispersive X-ray microanalysis (EDXMA). The experiment was conducted on cuttings grown in nutrient solutions with an increasing Zn concentration gradient (0.001–10mM). Biomass partitioning and Zn uptake were affected by the metal treatments, showing organ- and tissue-dependent responses. In particular, Zn accumulated in old leaves and moved from shoot to root as the Zn concentration in the growth medium increased. At the highest treatment concentration (10mM), Zn was preferentially localized in photosynthetic tissues of shoots, and in epidermis and cortex tissues of roots. Gas exchange and chlorophyll measurements showed impairments in leaf biochemistry rather than in stomatal function. Modifications in foliage area, stomatal density and leaf layer thickness were investigated to reduce and/or compensate the negative effects of excess Zn on CO₂ assimilation. To counteract Zn toxicity, clone I-214 adopted different defense/tolerance mechanisms involving complex structural, physiological and biochemical processes, attributed to both Zn excluders and accumulators. This study demonstrates the advantages of combining cryo-SEM/EDXMA, gas exchange and chemical analyses for studying metal localization and structural as well as physiological responses in plants.