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The phylogeny of Chloridoideae (Gramineae) was inferred from parsimony analyses of DNA sequences from two genomes—the chloroplast trnL intron, trnL 3' exon, and trnL–F intergenic spacer, and the nuclear ribosomal internal transcribed spacer region (ITS1 + 5.8S + ITS2). Eighty species representing 66 chloridoid genera were sampled, including all but four of the native New World genera. Analyses of the individual and combined data sets were performed. The phylogenies were found to be highly congruent. Of the four tribes and seven subtribes of Chloridoideae sensu Clayton and Renvoize (1986) whose phylogenetic status could be tested with our taxon sample, only Orcuttieae and Uniolinae were monophyletic. The phylogenies suggested significant homoplasy in morphological traits, including inflorescence type, number of florets per spikelet, and number of lemma nerves. We propose a new classification based on the three main clades in the phylogenies—tribes Cynodonteae, Eragrostideae, and Zoysieae. The Eragrostideae clade is well resolved and supported and is further divided into three subtribes, Cotteinae, Eragrostidinae, and Uniolinae. Cynodonteae include most of the genera in our study, but the clade is poorly resolved. However, a clade formed of Muhlenbergia and nine other genera is present in both phylogenies and is well resolved and supported. A number of interesting, well-supported relationships are evident in the phylogenies, including Pappophorum–Tridens flavus, Tragus–Willkommia, and Gouinia–Tridens muticus–Triplasis–Vaseyochloa. Except for Bouteloua, no genus represented by multiple species proved to be monophyletic in the phylogenies.