Physarieae, a tribe of mustards with multiaperturate pollen, was investigated phylogenetically for the first time using comprehensive sampling and plastid molecular data. Phylogenetic analyses were employed to test the monophyly of the tribe, to investigate relationships of its seven genera, and to study the evolution of selected morphological traits. Character evolution was inferred using parsimony approaches for nine continuous and three discrete traits related to trichomes, fruits, seeds, and pollen. Two monophyletic clades were recovered within Physarieae: the DDNLS clade, which included five genera (Dithyrea, Dimorphocarpa, Nerisyrenia, Lyrocarpa, and Synthlipsis), and the PP clade, which comprised two genera (Paysonia and Physaria). Under this new phylogenetic context, multiaperturate pollen appeared to be a potential synapomorphy of the tribe. In addition, five continuous fruit and seed traits were identified as new potential synapomorphies for Physarieae. Our analyses also indicated that members of the DDNLS clade present more highly branched dendritic trichomes, narrower replums, longer fruits, and wider seeds, when compared to representatives of the PP clade. Moreover, ancestral state reconstructions of continuous traits in Physarieae showed that seeds became wider and fruits, replums, styles, and seed primary grooves became longer over time.