Ten broccoli inbred lines (N237, N244, N256, N279, N318, N364, N437, N614, N622, N628) which are highly homozygous and uniform were used for genetic diversity and genetic relationship analysis based on inter simple sequence repeat (ISSR) molecular markers. Meanwhile, six nutrients of curds in these materials were also tested. Results showed that the overall similarity values among ten inbred lines ranged from 0.557 9 to 0.852 6 with an average of 0.703 2. From the cluster analysis result, ten broccoli inbred lines were divided into two clades (A and B). And clade A was further separated into four sub-clades. One inbred line which was rich in vitamin C (N256) and three inbred lines which had good multiple characters (N237, N279 and N628) were screened.