Abstract:The burrowing nematode,Radopholus similis is a quarantine nematode pest in the world.Using the universal primer pairs,the ITS region were amplified and sequenced,the ITS length were 706 bp,after analysis and alignment with known ITS sequences of R.similis.One species specific primer pairs,named RsF1/RsR1,was designed to diagnosis of burrowing nematode,the specific fragment was 271 bp.Combined the primer pairs D2A/D3B,the duplex PCR diagnosis for R.similis were developed to distinguish populations of R.similis from other plant parasitic nematode species,only R.similis populations could yield specie speci-fic 271 bp fragment,the other plant parasitic nematodes could not yield species specific fragment.