Brassica napus (AACC) is a recent allotetraploid species evolved through hybridization between two diploids, B. rapa (AA) and B. oleracea (CC). Due to extensive genome duplication and homoeology within and between the A and C genomes of B. napus, most SSR markers display multiple fragments or loci, which limit their application in genetics and breeding studies of this economically important crop. In this study, we collected 3,890 SSR markers from previous studies and also developed 5,968 SSR markers from genomic sequences of B. rapa, B. oleracea and B. napus. Of these, 2,701 markers that produced single amplicons were putative single-locus markers in the B. napus genome. Finally, a set of 230 high-quality single-locus SSR markers were established and assigned to the 19 linkage groups of B. napus using a segregating population with 154 DH individuals. A subset of 78 selected single-locus SSR markers was proved to be highly stable and could successfully discriminate each of the 45 inbred lines and hybrids. In addition, most of the 230 SSR markers showed the single-locus nature in at least one of the Brassica species of the U's triangle besides B. napus. These results indicated that this set of single-locus SSR markers has a wide range of coverage with excellent stability and would be useful for gene tagging, sequence scaffold assignment, comparative mapping, diversity analysis, variety identification and association mapping in Brassica species.