The oilseed Brassica napus L. is typically grown for edible oil; once the oil has been removed, the resulting seed meal is high in protein and has potential to be used as a dietary protein for human consumption. Specifically, the seed storage protein cruciferin is a candidate for development into a protein ingredient for food processing given its functional properties. Therefore, increasing cruciferin content in B. napus can add value to the crop by enabling the meal to be marketed as a secondary product. Traditional breeding efforts to increase cruciferin content in B. napus are hindered by the lack of information on the genetic variation of the trait and its underlying molecular mechanisms. The research presented in this dissertation aims to address these knowledge gaps. In the first project of the dissertation, a phenotyping protocol for cruciferin quantification was developed. The Western blotting method employed a custom anti-cruciferin antibody and was optimized to quantify cruciferin in soluble seed protein in B napus. The second project in the dissertation aimed to explore the genetic diversity of cruciferin content across a collection of spring B. napus cultivars that spanned 38 years of variety development. Genetic variation was observed in the population and varied from 44% to 93% of a reference cultivar. With genetic variation present, the third project of the dissertation used an association mapping approach to identify single nucleotide polymorphism molecular markers that were associated with cruciferin content to facilitate marker-assisted selection for the trait. Molecular markers on chromosomes A06 and A07 were found to be associated with cruciferin content and were in linkage disequilibrium with genes that may serve a regulatory role on cruciferin accumulation. Finally, to elucidate potential molecular mechanisms that govern differential cruciferin accumulation, a transcriptomics approach was taken in the fourth project of the dissertation. Using developing seeds from a high- and low-cruciferin cultivar, RNA sequencing revealed differential expression of regulatory genes, rather than cruciferin biosynthesis genes. Taken together, the knowledge gained from this dissertation will facilitate the breeding improvement of cruciferin content in spring B. napus and add value to the crop.