Cytoplasmic male sterility is a general phenomenon in almost 200 species, and the interaction between chimeric genes in mitochondria and restorer genes in nucleus may be responsible for restoration of male fertility. Orf288 has been identified as a CMS-associated gene in the hauCMS line of Brassica napus and Brassica juncea; however, the restorer lines/genes have not been found yet. We therefore have successfully constructed two restorer lines in B. napus by extensive testcrossing and have mapped a major restorer gene Rfh to a physical distance of 94 kb on chromosome A03 by whole-genome resequencing and molecular markers. We found that the restorer line is indeed restored to male fertility at histological level. Comparative genomics and collinearity analysis between close relatives revealed that rearrangements and recombination may have happened and thus caused the production of Rfh or components of the restoration of fertility complex. Meanwhile, nuclear backgrounds with multiple loci and temperature were related to the variation and instability of restoration of fertility in three different populations. Our study provides new sights into the coevolution between restorer genes and CMS-associated genes as well as the cultivation of superior hybrids via molecular breeding.