Agavaceae subfamily Chlorogaloideae is composed of the North American genera Camassia, Chlorogalum, Hastingsia, and Schoenolirion, with many species occupying serpentine soils or other poor soils with unusual chemistries. The monophyly and intergeneric relationships of this group have not been rigorously assessed. We estimated the phylogeny of Chlorogaloideae using four chloroplast DNA regions: rpl16 intron and trnD-trnY-trnE-trnT, psbJ-petA, and trnS-trnfM spacers, with the goals of evaluating 1) the monophyly of Chlorogaloideae, 2) the monophyly of each genus and generic interrelationships, 3) the placement of Chlorogaloideae in Agavaceae, and 4) the history of adaptation onto serpentine soils. Maximum parsimony, maximum likelihood, and Bayesian analyses provided concordant estimates of the phylogeny supporting the monophyly of a clade consisting of Camassia, Chlorogalum, and Hastingsia, but suggest that Schoenolirion may be more closely related to Hesperoyucca and Hesperaloe. Each genus of Chlorogaloideae was found to be monophyletic except Chlorogalum, with C. parviflorum and C. purpureum forming a paraphyletic grade to other Chlorogalum, Camassia, and Hastingia. Ancestral character reconstructions employing parsimony, likelihood, and stochastic mapping suggest that serpentine tolerance was gained and lost multiple times in Chlorogaloideae with comparable rates of gains and losses with a small bias towards losses. We discuss the significance of the estimated phylogeny for the evolution of the distinctive bimodal karyotype of Agavaceae.