Variation in life history strategies is a fundamental question in evolutionary biology, and the cooccurrence of annual and perennial habits in Castilleja and Castillejinae provides the opportunity to study the evolution of plant life history in a phylogenetic context. Molecular phylogenetic analysis of two chloroplast (rps16 and trnL/F) and two nuclear ribosomal (internal and external transcribed spacers) DNA regions support the monophyly of subtribe Castillejinae (Orobanchaceae). A well-supported phylogeny of the six genera (Castilleja [∼180 spp.], Clevelandia [1 sp.], Cordylanthus [18 spp.], Ophiocephalus [1 sp.], Orthocarpus [9 spp.], and Triphysaria [5 spp.]) comprising the subtribe is presented, and morphological synapomorphies are identified for the major lineages recovered. Orthocarpus and Triphysaria are both monophyletic; Cordylanthus is biphyletic. Clevelandia and Ophiocephalus are derived from within Castilleja. The perennial Castilleja clade (∼160 spp.) is derived from a grade of annual taxa including Castilleja sect. Oncorhynchus (16 spp.), Cordylanthus, Orthocarpus, and Triphysaria. This suggests that the perennial habit evolved a single time from an annual ancestral lineage that persisted throughout the diversification of Castillejinae, contrary to classical interpretations of life history evolution in plants. Given the prevalence of polyploidy among perennial Castilleja species, perenniality may have played an important role in the origin and establishment of polyploidy in Castilleja.