Random amplified polymorphic DNA (RAPD) analysis was performed on common wheat Chinese Spring, H. villosa, addition lines of H. villosa chromosome in CS, substitution line 3V of H. villosa chromosome in Triticum aestivum. A genome specific polymorphic DNA segment from H. villosa, OPF02757, was obtained. On the basis of cloning and sequencing of OPF02757, two PCR primers were designed and a genome specific PCR marker for H. villosa was established. The PCR marker including 677 bp was localized on all the seven pairs of H. villosa chromosomes. The result of PCR amplification by the primers indicated that there was a specific band of 677 bp in the materials containing H. villosa Chromosome such as T. aestivum-H. villosa addition, T. aestivum-H. villosa substitution, T. aestivum-H. villosa amphidiploid, T. durum-H. villosa amphidiploid and H. villosum from different accessions, and there was no specific band of 677 bp if the materials did not contain H. villosa chromosome, such as T. aestivum, T. durum, Secale cereale, Hordeum vulgare, Thinopyrum elongatum, Thinopyrum intermedium. Therefore, the PCR maker of 677 bp is specific to H. villosa genome, and could be used as molecular marker for detection of chromosomes of H. villosa in wheat.