Premise of research. The polybotryoid fern clade is completely Neotropical and consists of Cyclodium, Maxonia, Olfersia, Polybotrya, and Polystichopsis. It has never received a detailed phylogenetic analysis. We performed such an analysis to examine the relationships among species and genera and to map the evolution of their morphological and anatomical characters.Methodology. Our study included 46 (77%) of the 60 species in the clade. It also included 37 outgroup species from 19 genera. We sequenced four plastid DNA markers (rbcL, rps4-trnS, trnG-trnR, and trnL-trnF) and analyzed the data with maximum likelihood and Bayesian inference. One anatomical and 11 morphological characters were mapped on the resulting phylogenetic trees using the criterion of maximum parsimony.Pivotal results. The polybotryoid clade was strongly supported as monophyletic, as were its component genera. Nearly all its species have long-creeping rhizomes. Polystichopsis was resolved sister to the other polybotryoid genera. Its monophyly is supported by the morphological synapomorphies of distichous phyllotaxy, long straightish white hairs on the leaves, and tuberculate perines. Two species currently classified in Arachniodes (Arachniodes macrostegia and Arachniodes ochropteroides) form a clade with Olfersia. No known morphological characters support this clade. Olfersia, however, is highly distinct from all other polybotryoids by the combination of its imparipinnate laminae, submarginal connecting vein, strong sterile-fertile leaf dimorphy, loss of indusia, and evolution of acrostichoid sori. Maxonia is defined by its terrestrial root-climbing habit and dimorphic sterile and fertile leaves. Cyclodium and Polybotrya were resolved as sister. The presence of peltate indusia is synapomorphic for Cyclodium. Polybotrya is defined by several morphological synapomorphies: a rhizome anatomy unique among dryopteroid ferns (each individual meristele is surrounded by a dark sclerenchymatous sheath), strong sterile-fertile dimorphy, and loss of indusia. A possible synapomorphy for Polybotrya is the terrestrial root-climbing habit. Within Polybotrya, anastomosing veins and round discrete sori have evolved more than once.Conclusions. This is the first phylogenetic analysis of the polybotryoid ferns. The clade was resolved as monophyletic, as were its genera. An unexpected result was that two species currently classified in Arachniodes (A. macrostegia and A. ochropteroides) were resolved sister to Olfersia. Most of the main clades of polybotryoids were supported by morphological and/or anatomical characters.