Abstract: Flavanone 3-hydroxylase (F3H) is a key enzyme in the flavonoid-biosynthetic pathway. To clarify the sequence characteristics and substrate-catalytic properties of F3H from Pinus yunnanensis, we screened two F3H genes (PyF3H1 and PyF3H2) from the transcriptome of P. yunnanensis, cloned their full-length open reading frames, and performed comprehensive bioinformatic analyses. Recombinant proteins were expressed and assayed in vitro to determine their catalytic activities toward potential substrates. The full-length ORFs of PyF3H1 and PyF3H2 are 1,026 bp and 1,092 bp, respectively, encoding 341 and 363 amino acids. Phylogenetic analysis indicates that both proteins are most closely related to those of Ginkgo biloba, Thujopsis dolabrata, Sequoia sempervirens and Cryptomeria japonica. HPLC analyses confirmed that PyF3H1 and PyF3H2 catalyze the conversion of naringenin to dihydrokaempferol, thereby participating in the biosynthesis of dihydroflavonols. Tissue-specific expression analysis further revealed divergent expression patterns: PyF3H1 is specifically and highly expressed in lateral buds and young leaves, whereas PyF3H2 is predominantly expressed in shoot apices and young leaves. This study elucidates the distinct sequence features and substrate specificity of PyF3H1 and PyF3H2, providing a scientific basis for further dissecting the regulatory network of dihydroflavonol biosynthesis in P. yunnanensis.