Abstract: To screen DNA barcodes suitable for the identification of walnut cultivars and related species that were not easily distinguished by morphology, so as to provide a new way for accurate identification of walnut cultivars and ensure the healthy development of walnut industry, 11 walnut cultivars and 5 relatives were used as test materials, DNA barcodes with high amplification efficiency and abundant variation sites among cultivars were selected by amplification, sequencing and sequence variation analysis in three chloroplast genes（rbcL, psbA-trnH, matK） and one nuclear DNA sequence（ITS1）. The results showed that, the success rates of amplification and sequencing of the four candidate DNA barcodes in 16 species（cultivars） were 100%. The rbcL sequences showed minimal differences among species（cultivars）, while the psbA-trnH sequences differed only among genera. matK and ITS1 sequences differed among species（cultivars）, and their similarities were 99.12% and 99.88%, with 18 and 8 base site variants, respectively. Different species（cultivars） clustered differently in the phylogenetic tree constructed based on matK sequence, which verified the accuracy of using matK to identify walnut cultivars and their relatives. Thus, the combination of matK+ITS1 barcode can effectively identify walnut cultivars and their relatives.