In order to study genetic diversity and relationships of MAGIC parents of Brassica oleracea ,and provide the basis of the hybridization experiments of different subspecies.The singlecopy genes were identified with bioinformatics and some intron sequences of them were used to construct the phylogenetic tree of 12 parents from 7 subspecies.We identified 7 930 single copy genes form whole Brassica oleracea genome and randomly chosen from them of different chromosomes to amplify and sequence their intron sequences,resulting in a total of 3 427 bp intron sequences.Phylogenetic analysis revealed that intron sequence method was suitable for relationship analysis in the different sub species of Brassica oleracea L., which were obviously divided into three Clades:the Clade Ⅰ of cauliflowers and broccoli,the Clade Ⅱof kales and the Clade Ⅲ of head cabbage,brussels sprouts,kohlrabi and cabbage mustard.What’s more,cauliflower and broccoli are closely related and Chinese kale ( B. oleracea var. alboglabra Bailey) belongs to B. oleracea.The intron sequences of singlecopy genes will provide robust results on relationships of MAGIC parents and theoretical reference for hybridization of MAGIC parents in future.