Ogura Cytoplasmic male sterility CMS and its corresponding nuclear fertility restorer gene, Rfo, have been introduced from radish to Brassica species by interspecific crosses. Rfo restores male fertility by altering the translational expression of Orf138, a mitochondrial gene, whose expression results in the male sterile phenotype. This system has been extensively investigated and breeding restorer lines for the Ogura CMS has become a major objective for hybrid seed production in many canola breeding programs. In this study, we have sequenced genomic clones of Rfo amplified from a canola restorer line R2000, licensed from INRA, France, and a Dow AgroSciences proprietary non-restorer line Nexera 705 using primers designed from the radish Rfo sequence (GenBank accession AJ550021). Sequence alignment revealed three paralogs of Rfo. Two of the paralogs were present in both R2000 and Nexera 705 but the third one was present only in R2000. These results suggested that the first two sequences could be the orthologs of Rfo already existing in the canola genome and the third one could be the radish Rfo introduced into canola. Based on the sequence differences between the restorer and non-restorer lines, Rfo allele-specific PCR markers were developed. We also developed a high throughput, Rfo allele-specific Invader assay through Third Wave Technologies. Linkage analysis revealed a co-segregation between the markers and the phenotypes for fertility restoration. These markers have been proved to be very useful for direct selection of Rfo alleles for fertility restoration during marker-assisted introgression of the Ogura restorer for hybrid development in canola.