SummaryPast studies have identified herbivory as a likely selection pressure for the evolution of hyperaccumulation, but few have tested the origin(s) of hyperaccumulation in a phylogenetic context. We focused on the evolutionary history of selenium (Se) hyperaccumulation in Stanleya (Brassicaceae).Multiple accessions were collected for all Stanleya taxa and two outgroup species. We sequenced four nuclear gene regions and performed a phylogenetic analysis. Ancestral reconstruction was used to predict the states for Se-related traits in a parsimony framework. Furthermore, we tested the taxa for Se localization and speciation using X-ray microprobe analyses.True hyperaccumulation was found in three taxa within the S. pinnata/bipinnata clade. Tolerance to hyperaccumulator Se concentrations was found in several taxa across the phylogeny, including the hyperaccumulators. X-ray analysis revealed two distinct patterns of leaf Se localization across the genus: marginal and vascular. All taxa accumulated predominantly (65-96%) organic Se with the C-Se-C configuration.These results give insight into the evolution of Se hyperaccumulation in Stanleya and suggest that Se tolerance and the capacity to produce organic Se are likely prerequisites for Se hyperaccumulation in Stanleya.