Rapeseed (Brassica napus L.) is one of the most important oilseed crops worldwide, and the development of rapeseed varieties with high-quality oil is a long-term breeding goal. Reducing the content of palmitic acid, the main saturated fatty acid in rapeseed oil, can greatly improve oil quality. Here, a genome-wide association study (GWAS) and transcriptome-wide association study (TWAS) of seed palmitic acid content (SPAC) were performed using 393 diverse B. napus accessions. Four genes (BnaA08.DAP, BnaA08.PAA1, BnaA08.DUF106, and BnaC03.DAP) were identified by both GWAS and TWAS. The transcripts per million (TPM) values of these candidate genes at 20 and 40 days after flowering (DAF) were significantly correlated with SPAC in this association panel. Based on genetic variation in the candidate genes, four low-SPAC haplotypes were identified by combining candidate gene association analysis and haplotype analysis. Brassica napus accessions carrying low-SPAC haplotypes had lower SPAC than those carrying high-SPAC haplotypes without affecting seed oil content, seed protein content, or seed yield. Based on the functional single-nucleotide polymorphism (SNP) chrA08_9529850 (C/A) in the promoter of BnaA08.DUF106, a molecular marker (Bn_A8_SPAC_Marker) was developed that can be used to facilitate breeding for low SPAC in B. napus. Our findings provide valuable information for studying the genetic control of SPAC in B. napus. Moreover, the candidate genes, favorable haplotypes, and molecular marker identified in this study will be useful for breeding low-SPAC B. napus varieties.