The New World ‘justicioid’ lineage (Acanthaceae: Acanthoideae: Justicieae) includes about 500 species and encompasses extensive variation in flower shape, size, and color along with remarkable diversity in anther, seed, and pollen mophology. Species richness of this lineage is due to the inclusion of the New World members of the polyphyletic genus Justicia in addition to five smaller Neotropical genera: Cephalacanthus, Clistax, Harpochilus, Megaskepasma and Poikilacanthus. Although previous studies have shown that the New World ‘justicioid’ lineage is monophyletic, none have focused specifically on relationships among these plants nor has the current infrageneric classification of Justicia for the New World been tested. This is the first phylogenetic study to include a dense sample of taxa from the New World ‘justicioid’ lineage. We use DNA sequence data for five plastid, nuclear ribosomal ITS, and one low copy nuclear region (8315 bp) for 134 in-group taxa to estimate the phylogenetic framework for the group, and test the current infrageneric classification of New World Justicia. Most of the currently recognized sections of New World ‘justicioids’ are not monophyletic. On the other hand, our phylogenetic results identify nine major clades, many of which are supported by a combination of structural traits. The results of this study provide a foundation for the placement of other species of New World Justicia that have yet to be sampled for phylogenetic analysis, as well as for future comparative studies of evolutionary processes and revisionary work on this diverse and complex lineage.