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Phylogenetic relationships in the grass family (Poaceae), with specific attention to the internal structure of subfamily Pooideae, are analyzed on the basis of nucleotide sequence variation in plastid-encoded genes (matK, ndhF, ndhH, and rbcL). The resulting phylogenetic hypothesis was examined with attention to the taxonomic distributions of two inversions and an insertion/deletion within ndhF, the absence of intron 10 of the nuclear gene GBSSI (waxy), and positions of the boundaries between the Short Single Copy (SSC) region and the neighboring Inverted Repeat (IR) regions of the plastid genome, relative to the endpoints of ndhF and ndhH, which span these boundaries in some taxa. The PACCAD clade is resolved, and extension of the 3'-end of ndhF from the SSC region into the IR region is interpreted as a synapomorphy of this clade. The BEP clade also is resolved, with Ehrhartoideae placed as the sister of a clade in which Bambusoideae and Pooideae are sister groups. The loss of GBSSI intron 10 is interpreted as a synapomorphy of Poeae s.l., which includes the traditionally defined tribes Poeae, Aveneae, and Hainardieae, and the results support a novel set of relationships among the tribes of Pooideae, including the placement of Brachypodieae, Bromeae, Triticeae, and Poeae s.l. within a clade for which a three-nucleotide inversion in ndhF is interpreted as a synapomorphy, while a six-nucleotide inversion in ndhF marks a clade that includes all sampled members of subtribe Aveninae within Poeae s.l.