To obtain polymorphic markers between parents, a modified RFLP-subtractive hybridization method was developed. The method uses restriction fragments of a parent as a driver, which were ligated to form large fragment DNA, for hybridization with tester DNA, and the driver/tester hybrid fragments are removed by using a PCR purification kit. The method was used to isolate polymorphic sequences between Zhenshan97B and a Rf-gene near-isogenic line ZSR11 with normal and CMS-WA cytoplasms, respectively. As a result, two polymorphic sequences, Bgl II-1 and Pst I-4, were obtained. Southern blot analysis indicated that the polymorphisms were associated with the normal and CMS-WA types of cytoplasm.