Abstract: Two different genotypes of Stylosanthes germplasm, S. guianensis cv. Reyan No.2 and S. guianensis cv. Reyan No.13 were used as material for studying the effects of concentrations of Mg²⁺, dNTPs, DNA polymerase, primers and DNA template on the SRAP-PCR reactions and optimizing the establishment of SRAP molecular marker system in Stylosanthes. The optimum system was established as follows: template DNA 40 ng, Mg²⁺ 2.5 mmol/L, dNTPs 0.2 mmol/L, polymerase 1 U, primer 0.3 μmol/L, the total reaction volume was 25 μL. The genetic diversity and relationships of nine Stylosanthes germplasms were analyzed by using this optimum system. Forty-eight primers were studied for an analysis of genetic diversity by SRAP in Stylosanthes germplasms. Fourteen effective primers selected from 48 primers combination were used for SRAP-PCR, and 150 of the 154 DNA fragments amplified, showed polymorphisms. The average binds from each primer was 11.0 and the average percentage of polymorphic bands was 97.36%. The Nei’s genetic similarity coefficient of the tested accessions ranged from 0.386 to 0.882 by software NTSYSpc 2.1 based on SRAP results, and the average Nei’s coefficient was 0.631, average the genetic distance(GD) was 0.369. Based on the presence of bands, nine Stylosanthes germplasm were classified into there major groups by UPGMA cluster analysis, group Ⅰ, group Ⅱ and group Ⅲ. Group Ⅰ included S. hamata cv. Verano, and group Ⅱ included S. scabra cv. Seca. Group Ⅲ included S. guianensis cv. Reyan No.5, S. guianensis cv. Reyan No.10, S. guianensis cv. Reyan No.13, S. guianensis cv. White cook, S. guianensis cv. Reyan No.2, S. guianensis cv. Graham and S. guianensis tardio CIAT1283. This research should provide a scientific basis at the molecular level for further study and the application of nine Stylosanthes germplasms. The most of the varieties with relative relationship in their pedigrees and similar biological characteristics were clustered into the same group.