To assess the effectiveness of the new SCoT molecular marking technique in Saccharum spontaneum L., 157 Offsprings of the crosses between Saccharum spontaneum (GXS85-30) and Saccharum spontaneum(GXS87-16) were analyzed by SSR, AFLP and SCoT molecular marking technique respectively. Comparatively, SCoT molecular marking technique was better than SSR molecular marking technique in amplification of polymorphic DNA, and had better segregation marker than AFLP molecular marking technique. These results demonstrated that SCoT molecular marking technique had potential application prospects in genetic analysis of Saccharum spontaneum L..