Abstract: To characterize the flavonol synthase（ArFLS）gene family and its potential regulatory features in Anoectochilus roxburghii, a genome-wide bioinformatic analysis was conducted to investigate the physicochemical properties, chromosomal distribution, intragenomic synteny, conserved motifs, phylogenetic relationships, and promoter cis-acting elements of ArFLS family members. Four ArFLS genes were identified in the A. roxburghii genome and were located on Chr08A, Chr08B, Chr08C, and Chr08D, respectively. The encoded proteins comprised 328–377 amino acids, with molecular weights of 36.69–42.45 kDa and theoretical isoelectric points of 4.74–5.09. Subcellular localization prediction indicated that all four ArFLS proteins were localized in the cytoplasm. The four ArFLS genes showed a conserved distribution pattern among homologous chromosomes and exhibited syntenic relationships. Their encoded proteins shared similar conserved motifs and structural features, and all four proteins clustered in Group C based on phylogenetic analysis. Promoter analysis revealed that all four ArFLS genes contained cis-acting elements associated with light responsiveness, phytohormone responses, stress responses, and growth and development. These findings provide a basis for further investigation of the potential functions of the ArFLS gene family in flavonol biosynthesis and stress responses in A. roxburghii.