Flavonoids including anthocyanins provide flower and leaf colors and other derivatives that play diverse roles in plant development and interactions with the environment and dihydroflavonol 4-reductase (DFR) is part of an important step in the flavonoid biosynthesis pathway of anthocyanins. This study characterized 12 DFR genes of Brassica rapa and investigated their association with anthocyanin coloration, cold and freezing tolerance in several genotypes of B. rapa. Sequences of these genes were analyzed and compared with DFR gene sequences from other species and a high degree of homology was found. Constitutive expression of them in several pigmented and non-pigmented lines of B. rapa showed a correlation with anthocyanin accumulation only for BrDFR8 and 9. Conversely, BrDFR genes also showed responses to cold and freezing stress treatment in B. rapa. BrDFRs were also shown to be regulated by two transcription factors, BrMYB2-2 and BrTT8, contrasting with anthocyanin accumulation and cold and freezing stress. Thus, the above results suggest the association of these genes with anthocyanin biosynthesis and cold and freezing stress tolerance and might be useful resources for development cold and/or freezing resistant Brassica crops with desirable colors as well. The findings presented here may also help explore the molecular mechanism that regulates anthocyanin biosynthesis and its response to abiotic stress at the transcriptional level in plants.