The factors that affccting the ISSR(inter-simple sequence repeat)result of Astragalus sinicus.L. We studied and selected 33 ISSR primers which can amplify bands from the DNA of Astragalus sinicus.L.and the 33 ISSR primers all from the ISSR primers published by Columbia University. Bysetting the temperature gradient,the available primers’best annealing temperature are explored.The effect of the five main reaction elements(Mg2 、 TaqDNApolymerase、dNTP、 template and primer)on ISSR-PCR were all optimized by combining single factor experiments and othogonal test methed .then we establish the best ISSR-PCRreaction system.The best ISSR-PCR reaction system of Astragalus sinicus. is 25μl reaction system containting the DNA template 50.00ng,Mg2 concentration 2.00mmol/L，Taq enzyme dosageis 1.0U，dNTPs concention is 0.25mmol/L,primers0.20μmol/L, 2.5μL 10譩uffer. The seleted ISSR primers and annealing temperature is widespread and representative in Astragalus sinicusISSR-PCRmarker research and the result could provide the basis for the analysis of diversity , protection ;exploitation of SSR primers of Astragalus sinicus.