Abstract: The nrDNA-ITS sequence was used to investigate the genetic diversity and genetic relationship of 10 Lycium L. species. After using improved method of CTAB to extracted genomic DNA from the leaves of Lycium L., the authors set synthetic ITS4 and ITS5 as primers to amplify the nrDNA-ITS region and sequence target fragment in those DNA at the same time, then analyzed the sequencing result through cluster analysis. The results were as follows: the ITS length in those 10 Lycium L. species was from 582 bp to 656 bp, and there were 560 conservative sites (C), 95 variable sites (V), which included 70 parsimony-informative sites (Pi) and 25 single nucleotide mutation sites (S); the length of 5.8S sequence were all 154 bp, and there were 136 conservative sites (C), 18 variable sites (V), which included 18 parsimony-informative sites (Pi) and 6 single nucleotide mutation sites (S). Those 10 sequences cluster figures of 10 Lycium L. species were classified into 2 categories and 5 groups apparently, the authors found that the‘0901’and‘Mengqi 1’were in the same branch, and‘Ningqi 3’and‘Ningqi 7’were also in the same branch, both two groups had the closest genetic relationship; meanwhile,‘Ningqi 5’and Lycium ruthenicum Murray from Nuomuhong Qinghai were in different branch, which had the furthest genetic relationship. The authors had already uploaded all 10 ITS sequences of Lycium L. species to the NCBI, and the NCBI accession numbers were from KJ189761 to KJ189770 accordingly. This study had sequenced and analyzed the ITS sequences of 10 Lycium L. species, and the cluster results indicated the genetic relationship and difference among them, which would lay theoretical foundation for future study in genetic diversity and system development of Lycium L.