Random amplified polymorphic DNA (RAPD) analysis was performed on common wheat of Chinese Spring, addition lines of H. villosa chromosome in CS and H. villosum from different accessions with 100 random 10-base primers. A chromosome-specific polymorphic DNA segment for H. villosa, OPF02(750), was obtained from all addition lines of H. villosa chromosome in CS and H. villosum which belong to different accessions. The result amplified by primer OPF02 of all addition lines of H. villosa chromosome in CS indicated that all the seven pairs of H. villosa chromosomes contain OPF02(750) segment. There was no OPF02(750) in all Triticum aestivum and T. durum tested. Using OPF02, We confirmed that NAU302, an addition line of H. villosa chromosome 3V, had lost its chromosome 3V of H. villosa. Therefore, OPF02(750) is specific to chromosomes of H. villosa, and could be used as a molecular marker for detection of chromosome of H. villosa in wheat.