The Commelinaceae are a pantropical family of monocotyledonous herbs. Previous phylogenies in Commelinaceae have emphasized sampling among genera. We extended this previous work by sampling multiple species within some of the largest genera of Commelinaceae (especially Commelina and Tradescantia, and also including Callisia, Cyanotis, Gibasis, and Murdannia), and by sequencing noncoding regions both of the nuclear ribosomal DNA region, 5S NTS, and the chloroplast region, trnL-trnF. We generated a phylogenetic hypothesis for 68 Commelinaceae that partially tests previous morphological, taxonomic classifications. We found little evidence for conflict between nuclear and chloroplast regions for Tradescantia, Murdannia, and Callisia, and some evidence for conflict between the two regions for Commelina, though conflicting regions of the phylogeny were only weakly supported by bootstrap analyses. We found subtribe Tradescantieae to be paraphyletic, consistent with an rbcL study, though with a different topology than that produced by rbcL. In addition, subtribe Commelineae was monophyletic with strong support. We found Callisia to be polyphyletic, consistent with some previous molecular phylogenetic studies, and we found Tradescantia, Gibasis, Cyanotis, Commelina, and Murdannia, to be monophyletic. The molecular phylogenies presented here generally supported previous taxonomic classifications.