Abstract: Glucosinolates are amino acid-derived secondary metabolites that act as chemical defense agents against pests. However, the presence of high levels of glucosinolates severely diminishes the nutritional value of seed meals made from rapeseed (Brassica napus L.). To identify the loci affecting seed glucosinolate content (SGC), we conducted genome-wide resequencing in a population of 307 diverse B. napus accessions from the three B. napus ecotype groups, namely, spring, winter, and semi-winter. These resequencing data were used for a genome-wide association study (GWAS) to identify the loci affecting SGC. In the three ecotype groups, four common and four ecotype-specific haplotype blocks (HBs) were significantly associated with SGC. To identify candidate genes controlling SGC, transcriptome analysis was carried out in 36 accessions showing extreme SGC values. Analyses of haplotypes, genomic variation, and candidate gene expression pointed to five and three candidate genes in the common and spring group-specific HBs, respectively. Our expression analyses demonstrated that additive effects of the three candidate genes in the spring group-specific HB play important roles in the SGC of B. napus.