In order to develop and use the characteristic of mulberry varieties, twelve mulberry varieties were analyzed on genetic relationship and other three parameters, such as ITS, TrnL-F, rps16 nucleotide sequence length, G+C content and genetic distance. The tested mulberry varieties were Long sang, Long xu sang, Jian chi (high quality of mulberry), Xian feng chang sui sang (Guo sang), Qin zhou chang guo sang (Guo sang), Xiao guan sang (high quality of mulberry), Shen nong hua sang♀(Mu cai sang), Sri Lanka NO.1(Yi bu cheng yuan sang), Ya’an bai sang (Guo sang), Xinjiang bai sang (Medicine mulberry), Guangdon “Da shi” guo sang and Hubei meng sang NO.4(ecological mulberry). Experimental results: ITS region(including 5.8S) length, 576-590 bp, G+C%, 59.38-60.17; TrnL-F region(including TrnL introns), 920-923 bp, G+C%, 34.02-34.24; rps16 intron, 929-947 bp, G+C%, 32.51-33.26. The genetic distance of Jian chi (high quality of mulberry), Xian feng chang sui sang(Guo sang), Qin zhou chang guo sang(Guo sang), Xiao guan sang(high quality of mulberry), Shen nong hua sang ♀(Mu cai sang) from other Morus materials respectively is 0.2-0.3, 0.1-0.4, 0.1-0.2, 0.0-0.3. Long sang (Yuan lin sang), Sri Lanka NO.1(Yi bu cheng yuan sang), Ya’an bai sang(Guo sang), Xinjiang bai sang (medicine mulberry), Guangdong “Da shi”guo sang and Hubei meng sang with other genetic distance was very near, most of the genetic distance was 0. Mrbayes analysis showed that the length of bifurcation diagram was 191, the consistency index(CI) was 0.9895, the retention index(RI) was 0.9429, the consistency index adjusted(RC) was 0.9330. The length of sequence alignment was 2516 bp, among them, the invariant sites was 2330 bp, the variable non informative sites was 171 bp, the information site was 15 bp. The bifurcation diagram would first branch broussonetia papyrifera separated, which was a outgroup, while Xin jiang hei sang separated. Xian feng chang sui sang, Li chuan chang sui sang, Qin zhou chang guo sang were seperated respectively. Chuan sang, Hua sang, Nai sang, Shengnongjia hua sang♀(Mu cai sang), Mao ye nai sang belonged to a branch of mulberry. Guangdon “Da shi” guo sang, Si chuan bai sang (Guo sang) belonged to a branch of mulberry, Sri Lanka NO.1(Yi bu cheng yuan sang), Hubei meng sang NO.4(ecological mulberry), Long sang(Yuan lin sang), Xin jiang bai sang(medicine mulberry) belonged to a branch of mulberry. Long xu sang(Yuan lin sang), Jian chi(high quality mulberry), Xiao guan sang(high quality mulberry) was separately as a branch. Using the three segments(ITS, TrnL-F, rps16) to analyse system evolution of mulberry, the disadvantage of which is insufficient information site, these three pieces can be used to preliminary determine the genetic relationship, then choose other ways to analyse further.