DNA sequences were generated for the chloroplast gene ndhF to address phylogenetic relationships among the major lineages of the Poaceae. Our primary objectives were: 1) to test the monophyly of the Bambusoideae s.l., which traditionally has been viewed as the ancestral lineage of the family, and 2) to resolve phylogenetic relationships between disparate elements of this subfamily and the other major grass clades. A total of 45 grass sequences and two outgroup sequences were analyzed using parsimony and distance-based phylogenetic reconstruction methods. Two tribes of neotropical herbaceous bamboos, the Streptochaeteae and Anomochloeae, are resolved as the most basal clade within the family, with a clade representing the pantropical Phareae, also regarded as herbaceous bamboos, as the next most basal lineage. These results confirm that certain elements within the Bambusoideae s.l. are basal within the family and contrast with previous hypotheses that the Pooideae represent a basal or near-basal branch. Exclusive of the basal lineages of herbaceous bamboos, the remainder of the family formed a monophyletic clade with two primary subdivisions, a weakly supported "BOP" clade (bambusoids, oryzoids, and pooids), and a strongly supported PACC clade (panicoids, arundinoids, chloridoids, and centothecoids). Each of the three lineages of the BOP clade is well supported. The Bambusoideae s.l. and Arundinoideae are polyphyletic, and the inclusion of Thysanolaeneae and Centotheceae in a more broadly defined Panicoideae is supported. The Pooideae s.l. and Chloridoideae are resolved as monophyletic. The ancient evolutionary divergence within the Poaceae and the other relationships suggested by the phylogenetic analyses indicate that reevaluation of intrafamilial classification is necessary.