Petal size determines the value of ornamental plants, and thus their economic worth. However, the molecular mechanisms controlling petal size remain unclear in most non-model species. To identify quantitative trait loci and candidate genes regulating petal size in rapeseed (Brassica napus), we performed a genome-wide association study (GWAS) using data from 588 accessions over three consecutive years. We detected 17 significant single nucleotide polymorphisms (SNPs) associated with petal size, with the most significant SNPs located on chromosomes A05 and C06. A combination of GWAS and transcriptomic sequencing based on two accessions with extreme differences in petal size identified 11 differentially expressed genes (DEGs) that may control petal size variation in rapeseed. In particular, BnaA05.RAP2.2 homologous to RAP2.2 in rapeseed may be a critical gene negatively influencing petal size through the ethylene signaling pathway. In addition, a comparison of petal epidermal cells indicated that petal size differences between the two extreme accessions were determined mainly by cell number differences. Finally, we propose a preliminary model for the control of petal size in rapeseed. Our results provide insights into the genetic mechanisms regulating petal size, and also lay the foundation for a better understanding of petal development in plants.