Abstract: To investigate the biological function of flavonol synthase (FLS) genes in tobacco, members of the NtFLS gene family were identified from common tobacco variety K326, and bioinformatic analyses of gene structure, phylogenetic evolution, protein structure, etc. were performed. At the same time, the expression patterns of all NtFLS members in different tissues of tobacco were determined, as well as the expression levels of NtFLS1 and NtFLS2 genes under different hormonal and abiotic stress conditions. The results showed that: 1) The common K326 tobacco genome contained 9 NtFLS genes, all of which encode proteins with conserved 2OG-FeII_Oxy and DIOX_N domains belonging to the 2-oxoglutarate-dependent dioxygenase (2-ODD) superfamily. Among them, NtFLS1 and NtFLS2 proteins contained specific sequences of the FLS family and were closely related to NtomFLS in Nicotiana tomentosiformis and NsylFLS in Nicotiana sylvestris, respectively. 2) During the flowering stage, the expression levels of NtFLS1 and NtFLS2 genes in different tissues were significantly higher than those of the other 7 NtFLS genes, and showed higher expression levels in buds, stamens, and pistils, indicating that NtFLS1 and NtFLS2 had higher transcriptional activity and might play important regulatory roles in the development process of tobacco stamens and pistils. 3) The promoters of NtFLS genes contained multiple hormone and abiotic stress response elements, and the expressions of NtFLS1 and NtFLS2 genes were significantly regulated by multiple hormones and abiotic stress, indicating that NtFLS1 and NtFLS2 might be involved in regulating the response of tobacco to hormones and abiotic stress.