The anatomical structure of needles may reflect the functional status of Scots pine (Pinus sylvestris L.) seedlings at early ontogenetic stages. The study aimed to determine needle morphometric traits in seedlings of different clonal origin and to assess their variability. Seedlings of three clones were selected for analysis: D1 (slow-growing), D21, and D22 (fast-growing). Anatomical parameters were examined using light and fluorescence microscopy followed by digital image analysis. Significant interclonal differences were found in the main needle structures. In particular, needle length in fast-growing clones D21 and D22 exceeded that of slow-growing clone D1 by 3.5- to 3.7-fold and the control by 35-38% (p < 0.05). Cuticle thickness in fast-growing clones was, on average, 40-70% greater than in the slow-growing clone. Epidermal cell area increased by 50-70%; hypodermis development was marked by an increase in cell layer number from one to three; and plicate mesophyll width increased by 60-75%. For the vascular tissues, a 1.8- to 1.9-fold increase in vascular bundle area was recorded, while resin duct diameter exceeded the corresponding values of the slow-growing clone by 70-120%. 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 results indicate that seedlings with superior biometric performance exhibit a more differentiated needle anatomy compared to slow-growing variants. The observed correspondence between growth parameters and morpho-anatomical needle traits may reflect differences in the functional status of seedlings of different clonal origin. Pending further research on plant responses to stress factors, the identified morpho-anatomical traits could potentially serve as indirect indicators of enhanced seedling resilience to adverse environmental conditions.