Barleria is a genus of~300 species of herbs and shrubs in Africa and Asia, with highest diversity in tropical East Africa and southern Africa. The genus is part of Barlerieae (Acanthaceae), a lineage that is distinguished from other Acanthaceae by quincuncial corolla aestivation. Unequivocal synapomorphies have yet to be identified for Barleria. However, among Barlerieae, species of Barleria can be recognized by the combination of 4-partite calyces with the anterior and posterior lobes usually larger than the two lateral lobes, pollen with a coarsely reticulate (honeycomb) exine and the corolla limb variously subactinomorphic to strongly zygomorphic but never markedly bilabiate with a hooded upper lip. All Barleria that we have observed also have filaments that twist through 180°and cross just distal to the synstapetal zone. Barleria species have previously been classified into two subgenera and seven sections based on differences in leaf axil spine systems; configuration of the corolla, androecium and stigma; and morphology of the capsule and seed. We tested the subgeneric classification of Barleria by sampling 53 Barleria species and 9 outgroup species for three plastid intergenic spacers (trnS-G, ndhF-rpl32-trnL (UAG) ) and the nuclear region nrITS. We found a monophyletic Barleria and support for the two currently recognized subgenera; four of seven currently recognized sections are monophyletic but one of these four, sect. Fissimura, is nested within other sections. Species of sect. Fissimura unexpectedly resolved in subg. Barleria with strong support, although previous classifications placed the section in subg. Prionitis. We propose recognizing two subgenera (i.e., Barleria, Prionitis), the latter comprising three sections (i.e., Prionitis, Somalia, Stellatohirta). Barleria encompasses remarkable morphological diversity; we used an ultrametric tree to examine the evolution of nine morphological traits used to define infrageneric taxa in previous classifications. All characters evolved homoplasiously, but a few states (e.g., woody capsule septa) are supported as synapomorphies, and combinations of characters can be used to delimit subgenera. Members of subg. Barleria have 4-seeded capsules that lack a prominent beak, with a transition to 2-seeded capsules in the clade that corresponds to sect. Fissimura. In contrast, members of subg. Prionitis have 2-seeded capsules (reversed to 4-seeded in most members of one clade of sect. Somalia) that generally have a prominent beak. Additionally, we surveyed pollen morphology for 15 species sampled from across the classification. Pollen is consistently tricolporate with a coarse honeycomb exine. We provide an infrageneric classification and key to infrageneric taxa of Barleria.