To evaluate the monophyly of subtribe Pleurothallidinae (Epidendreae: Orchidaceae) and the component genera and to reveal evolutionary relationships and trends, we sequenced the nuclear ribosomal DNA internal transcribed spacers (ITS1 and ITS2) and 5.8S gene for 185 taxa. In addition, to improve the overall assessments along the spine of the topology, we added plastid sequences from matK, the trnL intron, and the trnL‐F intergenic spacer for a representative subset of those taxa in the ITS study. All results were highly congruent, and so we then combined the sequence data from all three data sets in a separate analysis of 58 representative taxa. There is strong support in most analyses for the monophyly of Pleurothallidinae and in some for inclusion of Dilomilis and Neocognauxia of Laeliinae. Although most genera in the nine clades identified in the analyses are monophyletic, all data sets are highly congruent in revealing the polyphyly of Pleurothallis and its constitutent subgenera as presently understood. The high degree of homoplasy in morphological characters, especially floral characters, limits their usefulness in phylogenetic reconstruction of the subtribe.