We used DNA sequence data from five genic regions (nrlTS; chloroplast trnL-F, trnT–L, rps16, trnS–G) to study phylogenetic relationships of the Tetramerium lineage (Acanthaceae: Justicieae). From a sample of 70 species (representing 25 genera) previously affiliated with the Tetramerium lineage, 68 are included therein. Our analyses excluded Papuasian Calycacanthus and Neotropical Streblacanthus monospermus from the Tetramerium lineage; however, two species described in Justicia (J. gonzalezii and J. medranoi) and a Malagasy species of uncertain generic affinities are nested within the lineage. A monophyletic Tetramerium lineage consists of 23 currently recognized genera with at least 168 species, more than 70% of which occur in the New World. Old World Chlamydocardia and Clinacanthus are serially sister to all other members of the lineage. Other Old World taxa consist of: Ecbolium clade (all sampled species of Ecbolium plus Malagasy Papulina richardii), Megalochlamys clade (Megalochlamys, Trichaulax and the unidentified Malagasy species), and two isolated taxa (Angkalanthus and Chorisochora). All analyses strongly support monophyly of the New World Tetramerium lineage. The basal dades of New World plants, all with nototribic flowers, are: 1) the taxonomically heterogeneous but palynologically consistent Mirandea clade, and 2) the Pachystachys clade the South American Anisacanthus clade. The second is sister to all other NW plants, referred to here as the core Tetramerium lineage. We recognize five clades within the core Tetramerium lineage related as follows: (Henrya clade (Carlowrightia parviflora clade (North American Anisacanthus clade (core Carlowrightia clade Tetramerium)))). Macromorphological synapomorphies are unknown for the Tetramerium lineage and for many of its constituent clades. However, we propose sternotribic flowers as synapomorphic for the core Tetramerium lineage, and flowers with the lower-central lobe of the corolla modified into a keel as a synapomorphy for a lineage consisting of Tetramerium and the core Carlowrightia clade. Palynological characters provide putative synapomorphies for some clades (e.g. Ecbolium clade, Mirandea clade) and autapomorphies for several species (e.g. Mexacanthus mcvaughii, Trichaulax mwasumbii). An Old World origin is postulated for the Tetramerium lineage, and we posit a single dispersal event to America and subsequent extensive radiation there, especially in arid zones of Mexico and adjacent regions. Taxonomic implications of our results are extensive. Notably, many traditionally recognized genera