Biocontrol fungus Pochonia chlamydospora ZK7 are highly effective in the biological control against root-knot nematodes infecting tobacco. To obtain the specific molecular marker of P. chlamydospora ZK7, distinct and reproducible molecular fingerprints of 97 isolates of different fungi, including biocontrol fungus P. chlamydospora ZK7, and some related and common soil fungi were amplified using the primers of random amplified polymorphic DNA (RAPD), intergenic spacers (IGS), enterobacterial repetitive intergenic consensus (ERIC), repetitive extragenic palindromic (REP) and a subunit of the Box element (BOX). Two specific DNA fragments of biocontrol fungus ZK7 were obtained upon amplification of genomic DNA of them with the random primers OPL-02 and OPD-05. Two amplified DNA fragments of 1200 (Vc1200) and 2000 (Vc2000)bp were diagnostic for P. chlamydospora ZK7, which clearly distinguishing them from 96 related isolates of control strains respectively. These two RAPD DNA fragments were isolated from agarose gel and purified, and the extracted DNA was ligated into a pGEM→-T Easy Vector, E. coli DH5 a competent cells were transformed with ligated DNA. The transformants were selected with blue-white dot and their plasmid DNA was extracted to verify the presence of inserts by electrophoresis. The cloned fragments were sequenced, their GenBank Accession No. were AY265801 and AY265802 respectively. Two pairs of oligonuceotide SCAR-PCR primers for each cloned fragment were designed. Use of the primers in the PCR assay resulted in the amplification of DNA fragments of the same size as the cloned RAPD fragments from genomic DNA of P. chlamydospora ZK7 respectively, but not control strains. These two amplified fragment specifically were labelled with digoxigenin and recognized the corresponding fragment present in the genomic DNA of P. chlamydospora ZK7 in dot blotting. In classical polymerase chain reaction, with a series of dilution of P. chlamydospora ZK7 DNA, the limit of detection of two pairs of oligonuceotide SCAR-PCR primers was 10 and 1000 pg/µl respectively, and the limit detection of dot blotting were 0.1 µg/µl.