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European larch (Larix decidua Mill.) has been reported either as more tolerant or as more sensitive to drought than conifers with perennial leaves. Previous studies have revealed that Carpathian populations of European larch display a high genetic variability. A comparative study of the responses of these populations to drought stress at the seedling stage might allow the identification of drought tolerant genotypes and reliable drought stress biomarkers, which could be eventually used for the early detection of drought effects in larch, not only under control greenhouse conditions, but also in their natural stands. Growth responses were analyzed in larch seedlings from six Romanian Carpathian populations, submitted to one month of mild drought stress under controlled conditions. Levels of photosynthetic pigments (chlorophylls a and b, and carotenoids), osmolytes (proline and total soluble sugars), monovalent cations (Na+ and K+), and malondialdehyde (MDA) and non-enzymatic antioxidants (total phenolics and flavonoids) were compared with control treatments and between populations. Growth and the pattern of the biochemical responses were very similar in the six populations. Drought stress lead to stem length decrease in all population, whereas reduction of fresh weight of needles was significant only in one population (BVVC), and reduction of water content of needles in two populations (BVVC and GuHo). The optimal biochemical traits for an early detection of drought symptoms in this species is the increase-in most populations-of total soluble sugars, MDA, and total phenolic compounds, whereas K+ reduction was significant in all populations. Photosynthetic pigments remained unchanged, except for the Anin population where they were reduced under stress. Multivariate principal component and hierarchical clustering analyses confirmed the impact of drought in the growth and physiology of European larch, and revealed that the humidity of the substrate was positively correlated with the growth parameters and the levels of K+ in needles, and negatively correlated with the levels of MDA, total soluble sugars, total phenolic compounds, and flavonoids in needles.