Phylogenetic analyses of internal transcribed spacer (ITS), external transcribed spacer (ETS), and 5.8S gene sequences of 18S–26S nuclear rDNA from all 23 genera of Cichorieae with centers of diversity in North America (and Picrosia from South America) show that all but three of the genera (Glyptopleura, Krigia, and Phalacroseris) belong to a series of seven clades that are well supported by bootstrap values >90%. Phalacroseris, endemic to California, with a single species (P. bolanderi), is sister to a well-supported (>95% bootstrap) clade that includes all other principally North American genera (plus Picrosia). The seven clades with major support and their component genera are: 1) the Lygodesmia Clade: Chaetadelpha, Lygodesmia, and Shinnersoseris; 2) the Pinaropappus Clade: Marshalljohnstonia and Pinaropappus; 3) the Pyrrhopappus Clade: Picrosia and Pyrrhopappus; 4) the Microseris Clade: Agoseris, Microseris, Nothocalais, Stebbinsoseris, and Uropappus; 5) the Stephanomeria Clade: Munzothamnus, Pleiacanthus, Prenanthella, Rafinesquia, and Stephanomeria; 6) the Malacothrix 1 Clade: Atrichoseris and various species of Malacothrix; and 7) the Malacothrix 2 Clade: Anisocoma, Calycoseris, and various other species of Malacothrix. The rDNA sequence data provide < 80% bootstrap support for other, larger groups that combine two or more of the seven major clades, except for one uniting all 24 ingroup genera and one uniting the Lygodesmia Clade and Pyrrhopappus Clade. The present analysis shows that Malacothrix, a genus of 22 species, is not monophyletic. None of the clades corresponds precisely to a suprageneric taxon of Cichorieae proposed previously, although taxa constituting each clade belong to a common subtribe or subgroup in classifications by Bremer, Jeffrey, and Stebbins, with two to three exceptions. As a group, the 24 genera represent a single, major radiation of Cichorieae based in North America.