Sequence data from the nuclear ITS region of 118 species (152 accessions) were used to test the monophyletic status and interrelationships of four related tribes in the Brassicaceae: Anchonieae, Chorisporeae, Euclidieae, and Hesperideae. Both maximum parsimony and maximum likelihood analyses support the recognition of the tribes Hesperideae (unigeneric, Hesperis L.) and Chorisporeae (3 genera, including Chorispora R. Br. ex DC., Diptychocarpus Trautv., and Parrya R. Br.), whereas the Anchonieae and the Euclidieae each separate into two distinct and distant clades (designated here as Anchonieae I and II and Euclidieae I and II). The data also support the exclusion of eight genera from the latter four tribes, with appropriate tribal assignment given in parentheses: Aubrieta Adans. (Arabideae), Blennodia R. Br. (Camelineae), Erysimum L. (Camelineae), Goldbachia DC. (unresolved), Hesperidanthus (B. L. Rob.) Rydb. (Schizopetaleae), Notothlaspi Hook. f. (unresolved), Pseudocamelina (Boiss.) N. Busch (Thlaspideae), and Zuvanda (Dvorák) Askerova (unresolved). The genus Malcolmia R. Br. (ca. 35 species) was paraphyletic and divided into three tribal clades, providing support for the separate genera Malcolmia, Strigosella Boiss., and Zuvanda. The genera Anchonium DC., Desideria Pamp., Eremobium Boiss., Erysimum, Iskandera N. Busch, Matthiola R. Br., Morettia DC., Neotorularia Hedge & J. Léonard, Rhammatophyllum O. E. Schulz, Sisymbriopsis Botsch. & Tzvelev, Sterigmostemum M. Bieb., and Tetracme Bunge each fell within a single tribal clade but were not monophyletic.