The study aimed to determine needle morphometric traits in seedlings of different clonal origin and to assess their variability. The anatomical structure of needles may reflect the functional status of Scots pine (Pinus sylvestris L.) seedlings at early ontogenetic stages. Seedlings of three clones – D1 (slow-growing) and D21, D22 (fast-growing) – were examined using light and fluorescence microscopy with digital image analysis (Image-Pro Premier 10.0). Statistical analyses included one-way ANOVA with Tukey’s HSD post hoc test, principal component analysis (PCA), and partial least squares discriminant analysis (PLS-DA). Analysis of needle anatomical parameters in Scots pine seedlings confirms that fast growth (clones D21 and D22) is associated with certain anatomical features compared to the slow-growing clone D1. Fast-growing clones typically have a more developed vascular system (wider phloem and xylem) and better structural protection (thicker cuticle and resin duct sclerenchyma). However, the variability in certain parameters – notably plicate mesophyll thickness and resin duct diameter – suggests that fast growth may result from different combinations of anatomical characteristics and compensatory mechanisms. Fluorescence microscopy data confirmed higher tissue autofluorescence intensity in clones D21 and D22 compared to the slow-growing clone, suggesting differences in cell wall composition and structure, as well as in the content of fluorescently active cellular components. Multivariate analysis (PCA and PLS-DA) revealed clear clustering of the studied clones and identified plicate mesophyll width, phloem width, and resin duct diameter as the anatomical traits contributing most to interclonal differentiation. The identified morpho-anatomical traits can serve as early-stage selection criteria for Scots pine seedlings, potentially reducing dependence on prolonged field trials. This is the first quantitative characterisation of the needle morpho-anatomical trait complex in Scots pine clonal seedlings at early developmental stages.