Abstract: Flavanone 3-hydroxylase (F3H), flavonol synthase (FLS) and anthocyanin synthase (ANS) are members of the 2-oxoglutarate-dependent oxygenase (2-ODD) superfamily, which play important functions in plant flavonoid biosynthesis. In this study, 16 2-ODD genes involved in flavonoid biosynthesis pathway were screened and identified from safflower genome database using bioinformatics method. The length of the proteins encoded by these genes ranged from 283 (CtFLS2) to 442 aa (CtANS6), and the molecular weight ranged from 32 062.05 (CtFLS2) to 48 245.49 Da (CtANS6). The proteins encoded by these genes were hydrophilic. Phylogeny divided it into three subfamilies, two conserved residues, H-x-D-xn-H and R-x-S, were found in this gene family using conserved motifs analysis, the analysis of cis-acting elements in the upstream 2 000 bp region showed that these genes might be regulated by environmental factors such as light, temperature and plant hormones such as salicylic acid, methyl jasmonate and so on. The members of the safflower 2-ODD gene family involved in flavonoid biosynthesis pathway were analyzed by qRT-PCR, the result showed that the expression patterns of these members were different in diverse flowering period and leaf. This study can lay a foundation for further exploring the function of 2-ODD genes involved in flavonoid biosynthesis pathway in safflower.