S45AB, a recessive genic male sterile (RGMS) line, originated as a spontaneous mutant in Brassica napus cv. Oro. The genotypes of sterile (S45A) and fertile plants (S45B) are Bnms1ms1ms2ms2 and BnMs1ms1ms2ms2, respectively. In our previous studies, Yi et al. (Theor Appl Genet 113:643-650, 2006) mapped the BnMs1 locus to a region of 0.4 cM, candidates of which have been identified and genetic transformation is in progress. We describe the fine mapping of BnMs2 exploiting amplified fragment length polymorphism (AFLP) and amplified consensus genetic marker (ACGM) methodologies, and the identification of a collinear region probably containing BnMs2 orthologue in Arabidopsis thaliana. A near isogenic line (NIL) population S4516AB which segregated for BnMs2 locus was generated by crossing, allelism testing and repeated full-sib mating. From the survey of 1,024 AFLP primer combinations, 12 tightly linked AFLP markers were obtained and five of them were successfully converted into co-dominant or dominant sequence characterized amplified region (SCAR) markers. A population of 2,650 sterile plants was screened using these markers and a high-resolution map surrounding BnMs2 was constructed. The closest AFLP markers flanking BnMs2 were 0.038 and 0.075 cM away, respectively. Subsequently, an ACGM marker was developed to delimit the BnMs2 locus at an interval of 0.075 cM. We extended marker sequences to perform BlastN searches against the Arabidopsis genome and identified a collinear region containing 68 Arabidopsis genes, in which the orthologue of BnMs2 might be included. We further integrated BnMs2 linked AFLP or SCAR markers to two doubled-haploid (DH) populations derived from the crosses Tapidor x Ningyou7 (Qiu et al., Theor Appl Genet 114:67-80, 2006) and Quantum x No.2127-17 (available in our laboratory), and BnMs2 was mapped on N16. Molecular markers developed from these investigations will facilitate the marker-assisted selection (MAS) of RGMS lines, and the fine map and syntenic region identified will greatly hasten the process of positional cloning of BnMs2 gene.