Abstract: Now amplified fragment length polymorphism (AFLP) is one of the usest molecular marker methods,In this paper ,we used wheat rust fungus as materials.Through extracting DNA、the digestion f DNAof wheat rust fungus、PCR amplification and so on, we established the AFLP markers technical system for rust fungus：400ng quantity of DNA was digested with 5U EcoRI and 5U TrulI at 37℃for 3hours and then 65℃ for 3 hours,then added ligation mixture at 22℃ for 3hours and 16℃ for 10 hours；pre-selective amplification were set up 5μl restrictiom-ligatiom samples DNA,10uM pre-selective amplification primers each 1.5μl,PCR mixture 25μl,ddH2O 17μl ;selective amplification were set up DNA 5μl, selective amplification primers each 1μl, PCR mixture 10μl,ddH2O 3μl. These results provided an useful tool to research the molecular cloning of wheat rust fungus and other fungus disease and breeding assisted selection.