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Wild cherry (Prunus aviumL.) is a multi-purpose tree species with great ecological and economic importance for European forestry. Evaluating this species phenotypic diversity and quantitative traits characterization is of great importance to define its genetic resources conservation and breeding strategies. In this work, variations of physiological, biochemical, anatomical and morphological traits of one-year-old wild cherry seedlings were evaluated within and among populations to distinguish and characterize their phenotypic portfolio. We observed significant differences at the intra- and inter-population levels considering both biochemical and physiological leaf traits, whereas differences in morphological and anatomical traits were found to be significant only among half-sib lines within populations (i.e. intra-population level). With a multivariate approach, we explored the inter-population specificity and found out that the tiered approach spanning from organ morphology, across physiological scale, to the biochemical level gave out enough power to discriminate between different populations, and their acquisition and resource-use strategies. Moreover, stepwise discriminative analysis showed that radical scavenger capacity against 2,2’-azino-bis(3-ethylbenzothiazoline- 6-sulfonic acid) (ABTS•+) and water-use efficiency contributed to discrimination of studied populations to the largest extend. Lastly, our study highlights the robustness of certain functional traits, such as ABTS•+, water-use efficiency, net photosynthesis, total flavonoid content, width of stomata guard cell, and stomatal aperture length, which could be considered as a proxy to discriminate between wild cherry populations and assess phenotypic diversity.