Among Acanthaceae (ca. 4,000 spp.), Barlerieae (ca. 440 spp.) is a tribe that needs considerable further study. Barlerieae contain 13 genera, the two largest and most widespread of which are Barleria (ca. 300 spp., primarily paleotropical), and the pantropical Lepidagathis (ca. 100 spp.). Sub-Saharan Crabbea includes 14 species. The remaining ten genera, with ~25 species in all, are distributed across Latin America, South Asia, Africa, and Madagascar. Barlerieae present several taxonomic problems, and some species have intriguing biogeographic patterns (e.g., a few species, such as Lepidagathis alopecuroides, have amphi-Atlantic distributions). Many species in this group are important to tropical ecosystems and support a wide range of pollinators. In a recent reclassification of Acanthaceae, three subtribes have been proposed based on corolla and calyx characters: (1) Barleria, (2) Crabbea and allies, and (3) Lepidagathis and allies. Using a next-generation sequencing (NGS) approach (ddRADseq), I will: (1) estimate a well-sampled phylogeny of the tribe to test current taxonomy as well as proposed classifications, (2) test genera and sub-generic groups for monophyly, (3) identify morphological characters that support monophyletic groups, (4) examine patterns of biogeography and diversification, and (5) address conservation concerns and prospects in a phylogenetic context. A Maximum Likelihood phylogeny of 153 taxa was estimated in IQ-TREE with 1000 Ultrafast Bootstraps, using GTR+F+I+G4 model, based on an alignment of ddRAD 59,304 loci, resolving relationship among all thirteen currently recognized genera in Barlerieae. This group, which can be recognized by the developmental character quincuncial corolla aestivation, is monophyletic with our sampling. In addition, data suggest a major taxonomic revision of Lepidagathis is necessary.  Species of Lepidagathis are found in three clades: Lepidagathis s.s., including species with bithecous posticous anthers, species formerly recognized as Lophostachys + Lepidagathis with monothecous posticous anthers; and the widespread species Lepidagathis aloepecuroides. The phylogenetic relationships recovered here suggest each of the three clades may merit recognition at the generic level. In addition, L. aloepecuroides is found to be monophyletic with sampling across its range in Africa and South America. Multiple transcontinental disjunctions across the tribe have also been illuminated. This research will contribute to taxonomic revisions and updated keys that will aid in conservation efforts and provide insight into the evolutionary processes that shape biodiversity in this remarkable lineage.