Map-based gene cloning is now widely used to isolate genes of unknown products. After fine mapping of a target gene is completed, the key step to head forward is to construct series of contigs covering the target gene through chromosome walking. To approach the final goal of cloning a wide compatible gene of rice (S5 locus), the flanking G200 marker was used as a starting point to walk. Four positive clones ranging from 240 approximately 650 kb were obtained from the rice genomic YAC library (RGP, Japan). With the ends of YAC inserts isolated by inverse-PCR, a YAC contig of 3 cM was initially built. Next, chromosome walking was performed with the two distal ends of the contig and another seven positive YAC clones were screened out, leading to the contig extending to about 8 cM.