Vegetable crops can be significant sources of nutritionally important dietary carotenoids and Brassica vegetables are sources that also exhibit antioxidant and anticarcinogenic activity. The family Brassicaceae contains a diverse group of plant species commercially important in many parts of the world. The six economically important Brassica species are closely related genetically. Three diploid species (B. nigra, B. rapa, and B. oleracea) are the natural progenitors of the allotetraploid species (B. juncea, B. napus, and B. carinata). The objective of this study was to characterize the accumulation of important dietary carotenoid pigments among the genetically related Brassica species. The HPLC quantification revealed significant differences in carotenoid and chlorophyll pigment accumulation among the Brassica species. Brassica nigra accumulated the highest concentrations of lutein, 5,6-epoxy lutein, violaxanthin, and neoxanthin. The highest concentrations of beta-carotene and total chlorophyll were found in B. juncea. Brassica rapa accumulated the highest concentrations of zeaxanthin and antheraxanthin. For each of the pigments analyzed, the diploid Brassica species accumulated higher concentrations, on average, than the amphidiploid species. Brassicas convey unique health attributes when consumed in the diet. Identification of genetic relationships among the Brassica species would be beneficial information for improvement programs designed to increase carotenoid values.