Abstract: In this study, extract high quality genomic DNA from jute (Corohorus olitorius L) and establish a stable SRAP reaction system, including template DNA concentration, Mg2+ concentration, Taq DNA polymerase concentration, dNTPs concentration and forward and reverse primer concentration, using a population F2 progeny derived from a cross of glycanes thesia (wild species) and wild leaf jute (cultivated species). The results showed that the DNA isolated with modified CTAB method had good quality. The optimum SRAP reaction system could amplify high levels of polymorphism, good repeatability and clear band pattern. SRAP-PCR system (total volume of 25 l) was established as follows: template DNA 100 ng, forward primer 0.48 molL-1, reverse primer 0.48 molL-1, Mg2+ 2.8 mmolL-1, dNTPs 0.35 mmolL-1, Taq DNA polymerase 0.7 U. It showed that the optimized system can be applied in the SRAP analysis for Corohorus olitorius L., which provided technical support for the genetic linkage map construction in the future.