The recessive genic male sterility (RGMS) line 9012AB has been used as an important pollination control system for rapeseed hybrid production in China. Here, we report our study on physical mapping of one male-sterile locus (BnRf) in 9012AB by exploiting the comparative genomics among Brassica species. The genetic maps around BnRf from previous reports were integrated and enriched with markers from the Brassica A7 chromosome. Subsequent collinearity analysis of these markers contributed to the identification of a novel ancestral karyotype block F that possibly encompasses BnRf. Fourteen insertion/deletion markers were further developed from this conserved block and genotyped in three large backcross populations, leading to the construction of high-resolution local genetic maps where the BnRf locus was restricted to a less than 0.1-cM region. Moreover, it was observed that the target region in Brassica napus shares a high collinearity relationship with a region from the Brassica rapa A7 chromosome. A BnRf-cosegregated marker (AT3G23870) was then used to screen a B. napus bacterial artificial chromosome (BAC) library. From the resulting 16 positive BAC clones, one (JBnB089D05) was identified to most possibly contain the BnRf (c) allele. With the assistance of the genome sequence from the Brassica rapa homolog, the 13.8-kb DNA fragment covering both closest flanking markers from the BAC clone was isolated. Gene annotation based on the comparison of microcollinear regions among Brassica napus, B. rapa and Arabidopsis showed that five potential open reading frames reside in this fragment. These results provide a foundation for the characterization of the BnRf locus and allow a better understanding of the chromosome evolution around BnRf.