Physarieae is a small tribe of herbaceous annual and woody perennial mustards that are mostly endemic to North America, with its members including a large amount of variation in floral, fruit, and chromosomal variation. Building on a previous study of Physarieae based on morphology and ndhF plastid DNA, we reconstructed the evolutionary history of the tribe using new sequence data from two nuclear markers, and compared the new topologies against previously published cpDNA-based phylogenetic hypotheses. The novel analyses included ca. 420 new sequences of ITS and LUMINIDEPENDENS (LD) markers for 39 and 47 species, respectively, with sampling accounting for all seven genera of Physarieae, including nomenclatural type species, and 11 outgroup taxa. Maximum parsimony, maximum likelihood, and Bayesian analyses showed that these additional markers were largely consistent with the previous ndhF data that supported the monophyly of Physarieae and resolved two major clades within the tribe, i.e., DDNLS (Dithyrea, Dimorphocarpa, Nerisyrenia, Lyrocarpa, and Synthlipsis) and PP (Paysonia and Physaria). New analyses also increased internal resolution for some closely related species and lineages within both clades. The monophyly of Dithyrea and the sister relationship of Paysonia to Physaria was consistent in all trees, with the sister relationship of Nerisyrenia to Lyrocarpa supported by ndhF and ITS, and the positions of Dimorphocarpa and Synthlipsis shifted within the DDNLS Clade depending on the employed data set. Finally, using the strong, new phylogenetic framework of combined cpDNA + nDNA data, we discussed standing hypotheses of trichome evolution in the tribe suggested by ndhF.