In this study, through homologous gene cloning technology, three complete CDSs encoding dihydroflavonol 4-reductase(DFR), a key enzyme in the pathway of anthocyanin biosynthesis, were cloned from the corolla with white, red and blue color of petunia hybrid planted in Xinjiang respectively, and named PhDFR1, PhDFR2 and PhDFR3. Homology alignment indicated that the nucleotide similarity of the three DFRs is 97.79 %、96.59% and 97.99% with another DFRA gene from Petunia hybrida(GenBank access number:X15537). All these DFR CDSs encoded a polypeptide composed of 380 amino acid residues. The amino acid residues similarity of the three DFRs is 95.53 %、94.21% and 95.79% with DFRA protein(GenBank access number:CAA33544). DFRs of the three Petunia hybrida strains with different flower color contain a highly conserved NADP(H)-binding site and substrate specificity site which belong to NADB-Rossmann superfamily. They are stable proteins without signal peptide and are hydrophilic proteins probably located in cytoplasm. All of them have two transmembrane domains. alpha-helix and beta-sheet are primary secondary structural components of DFR. The beta-alpha-beta-alpha-beta structures forming Rossmann folding are symmetrically distributed on the whole. The phylogenyetic analysis of DFR proteins from different species revealed that three DFRs have closer relationship with the DFR from Dianthus caryophyllus than from the other plant species.