Abstract: To establish and optimize reaction system, the effects of different factors on random amplified polymorphic DNA (RAPD) reaction system in Sudangrass were studied and the fingerprinting of two hybrids to differentiate them were constructed. Genome DNA was isolated. The different concentration of Mg2+(1.0mmol/L, 1.5 mmol/L, 2.0 mmol/L, 2.5 mmol/L, 3.0 mmol/L), annealing temperature (34℃, 35℃, 36℃, 37℃, 38℃) and Tap DNA polymerase activity (0.6U, 0.8U, 1.0U, 1.2U, 1.4U) were designed for PCR amplification. The results were as follow: the optimization of a 20μl PCR system was coupled with the concentration of Mg2+2.0 mmol/L, Taq DNA polymerase activity 0.6U and the annealing temperature 36℃. The DNA fingerprinting of two hybrids was constructed by RAPD marker. Two special bands, A and B, can differentiate Wancao2 and Wancao3 in the fingerprinting.