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The aim of this study was to propose a non-destructive method for measuring Pinus sylvestris seedlings, to determine whether water stress would be evident in laboratory spectra of pines, to compare whether the response of pines would differ by ecotype, and to investigate whether two ecotypes of Pinus sylvestris could be distinguished using laboratory and image spectroscopy. For these purposes, hyperspectral images of seed orchards from August 2020 were processed and a three-month laboratory experiment was conducted, in which stress from water deficit was induced in two-year-old pine seedlings from the upland and hilly ecotypes. Spectral data were analysed using mixed statistical models, analysis of variance, principal component analysis, training of supervised pixel classifiers, vegetation indices, and linear regression. Based on the analyses, it was found that water stress can be detected in severely stressed Pinus sylvestris seedlings. The most sensitive spectral bands to water content were observed in the region between 1000-2500 nm. The initial response to water stress did not differ by ecotype, but a faster recovery was observed at the upland ecotype after the period of draught. The two Pinus sylvestris ecotypes were distinguished with high accuracy from both laboratory and image spectral...