The fatty acid desaturase genes are the main contributors to oleic acid content, and different alleles can result in different oleic acid contents in rapeseed oil. Hence, identification of allelic variation in is an extremely desirable breeding goal. By performing QTL mapping using 190 F lines genotyped by genome-wide single nucleotide polymorphism (SNP) markers assayed by the 60 K Infinium BeadChip Array, four quantitative trait loci (QTL) for C18:1 content were mapped on chromosomes A01, A05, A09 and C05 over 3 years in a population segregating for oleic acid content. Two genes on A05 and C05 were anchored within the QTL intervals, explaining 45-52 and 15-44% of the observed variation for C18:1 content. Sequence polymorphisms between the corresponding coding regions of the parental lines found two single-nucleotide polymorphisms (SNPs) in and , respectively, which led to the amino acid changes (C421T and G1073E) in the corresponding proteins. The mutation sites of and alleles were located within the second H-box and near the third H-box motif of the protein, respectively, and were found to be novel mutant alleles. Lines resulting from the combination of these two alleles contained up to 88% oleic acid in their seed oil, compared with 63% in wild-type controls. Two competitive allele-specific PCR (KASP) markers based on these two mutation sites were successfully developed and validated in segregating F populations. These markers will facilitate breeding for ultra-high seed oleic acid content in oilseed rape.