Hybrid sterility is the main barrier in utilizing the heterosis of subspecies in rice. A knowledge of the underlying molecular mechanism of the hybrid sterility will be useful for overcoming the barrier. In this research, the F1 pollen sterility locus, S-b, was mapped between SSR markers PSM8 and PSM202. To fine map the locus, one F2 mapping population of 3910 plants was developed using the near-isogenic lines of the locus. Ninety-seven recombinants between two markers were selected. Moreover, a series of markers, including two SSR markers, two InDel markers and four CAPS markers, were developed on the region. Linkage analysis showed that marker W4 was co-segregated with locus S-b, while makers A8 and A14 were located on the two sides of the locus with a distance of 0.026 and 0.038 cM, respectively. The markers were then integrated with the sequences of the clones of the region. Results showed that all the polymorphic markers were anchored on the three end-to-end jointed clones AC093089, AC079021 and AC134931. According to the physical information of the markers, locus S-b was finally delimited to a region of 27 kb between A8 and A14. Seven ORFs were identified on the region based on the annotation results of RiceGAAS system. These results laid the foundation for further cloning the gene.