Spontaneous male sterility is an advantageous trait for both producing hybrids and understanding the developmental process of the male reproductive unit in many crops. Lu et al. use a map-based cloning strategy together with comparative mapping to study the triallelic genetic male-sterile locus BnMs5 in Brassica napus, and delimit it to a 21-kb fragment of the A8 chromosome. Sequence analysis suggests that BnMs5 originates from the B. rapa MF2 subgenome, and may reside in a hotspot region of chromosomal evolution. This work paves the way for further cloning of BnMs5, and presents a powerful method for mapping loci in plants with complex genomic architecture.