Forty-eight representatives of 12 tribes attributed to the subfamily Pooideae s.l. of grasses (Monocots) have been studied by sequencing the more variable 3' end of the chloroplast ndhF gene. Six representatives from 5 different tribes of Poaceae (Oryzeae, Streptogyneae, Bambuseae, Arundineae, Phareae) and from Joinvilleaceae were used as outgroups. Phylogenetic analyses of the data render a monophyletic Pooideae clade and provide an evolutionary hypothesis for all its tribes (Poeae, Aveneae, Bromeae, Triticeae, Brachypodieae, Meliceae, Stipeae, Lygeae, Nardeae, Diarrheneae, Brachyelytreae, Phaenospermatae). The subfamily is composed of two subsets of tribes, a basal group that includes six old lineages or satellite tribes, in which Brachyelytreae is the first diverging branch followed by the Lygeae/Nardeae clade, and a group of six more recently evolved tribes (Pooideae s.s.), in which the divergence of Diarrheneae antedates that of Brachypodieae, sister group to the "core pooids." The character changes found within the sequenced nucleotide positions of the ndhF gene also have proven to be informative at lower hierarchical levels (genus and species) for some tribes.