Lycium is an important economic genus belonging to solanaceae. It is widely distributed in Northwest China and highly adaptable to the environment. In this study, SSR markers were developed based on the full-length transcriptome of Lycium variety ‘Ningqi 1’, followed by genotyping of 28 Lycium germplasm using a subset of polymorphic SSR markers. SSR loci were in silico identified using MISA software and subjected for primer pickup. Twenty-three SSR markers selected from 206 pairs of primers were used for genotyping and analyzing the genetic diversity of Lycium barbarum germplasm from 28 different distribution regions. Genetic coefficients were clustered by unweighted group average (UPGMA) method. A total of 240 alleles were amplified using 23 SSR markers with 4 to 17 alleles at each locus. The maximum Shannon information index was 1.177 to 2.487. The expected and observed heterozygosity ranged from 0.635 to 0.909 and 0.250 to 0.964, respectively. PIC ranged from 0.58 to 0.902. The genetic distance clustering analysis suggested four groups in 28 Lycium germplasm (similarity coefficient = 0.71). The Ningqi series germplasm belonged to group Ⅰ, the Tianjing series and Guangdong germplasm belonged to group Ⅱ, the Lycium ruthenicum germplasm belonged to group Ⅲ, and only Gouqi Island germplasm belonged to group Ⅳ. The total length transcriptomic sequence of Ningqi 1 could be used to develop SSR markers. Collectively, the full-length transcriptome sequences of Lycium are of interest to develop SSR markers, and these newly-developed SSR markers can be used for genetic diversity analysis of Lycium germplasm and molecular marker assisted breeding.