Using rapid amplification of cDNA ends (RACE) methods, a full length cDNA homologuous to CBF was cloned with two degenerate primers from Juglans regia L.. The complete cDNA sequence was designated as JrCBF and the sequence was submitted to GenBank (GenBank accession number JX875914). Sequence analysis showed that the nucleotide sequence of JrCBF was 879bp, encoding a protein with 214 amino acids. The real-time PCR technology was used to analyze the expression levels of JrCBF gene when seedlings were treated with cold stress. JrCBF gene expression was increased in 2 h after treated with low-temperature of 4 ℃and reached to a peak after 8 h. Under the condition of natural overwintering, JrCBF gene expression was first rise after falling, reaching to a peak at severe winter (in January). Furthermore, single nucleotide polymorphism was analyzed among 15 varieties. In the Juglans regia L. populations, 28SNPs and seven Insertion/Deletion (indel) were detected and two hot mutation regions existed. Haplotype analysis showed that 15 varieties were divided into nine haplotypes, and the haplotype diversity was 0.9238. The successful cloning of JrCBF gene from J. regia and confirming cold resistance target gene can provide a basis for cultivating cold resistance variety and for molecular marker assistant breeding.