Polyploidy is a major evolutionary process in flowering plants yet its effects on biotic interactions are still unclear. Whole genome duplication is expected to have impacts on floral traits and selfing propensity which can have cascading effects on pollination interactions. Since the vast majority of flowering plants rely on animal-mediated pollination, the degree of pollination generalization is also expected to play an important role in polyploid population establishment as well on the structure of their communities. Here we used herbarium specimens to assess the effects of polyploidy and mating system on floral traits and pollination niche of 40 Brassicaceae species. For each species, we obtained mating system (self-compatible=SC or self-incompatible=SI) and chromosome counts from the literature and inferred ploidy level (polyploid or diploid). Specifically, we investigated the influence of polyploidy and mating system on 1) flower size and 2) shape (evaluated using PCA analysis of flower tube, length, and width of the attractive portion of petals), 3) the frequency of stigmas with heterospecific pollen, and 4) the richness of heterospecific pollen morphotypes. Based on phylogenetically controlled models, we found an interaction effect of the polyploidy and mating system on flower size, with SI polyploid species having larger flowers than SC polyploids. Floral shape also changed with mating and ploidy but not significantly. With respect to the pollination niche, we only detected an effect of mating system. That is, SI species had more stigmas with heterospecific pollen and more heterospecific pollen morphs on average than SC species, regardless of their ploidy. Our results suggest that mating system moderated the influence of ploidy on morphological features of pollination generalization but that of ecological generalization is more variable.