Abstract Ophioglossaceae are a putatively ancient lineage of ferns in which the aerial portion of the plant is composed of a single leaf. The simplicity of foliar morphology has limited the number of characters available for constructing classifications and contributed to taxonomic difficulties at nearly every level of classification within the family. Analysis of plastid DNA rbcL sequences from 36 species representing the diversity of Ophioglossaceae supported the monophyly of the family. Intrafamilial relationships were examined using rbcL and trnL-F plastid DNA sequences and morphological data. Individual and combined analyses of the three data sets revealed two main clades within the family, here termed ophioglossoid and botrychioid. In the botrychioid clade, Helminthostachys was sister to a broadly defined Botrychium, within which Botrychium in the narrow sense of some authors and Sceptridium were sister. Botrypus was paraphyletic, with Botrypus virginianus sister to Botrychium plus Sceptridium, and with Botrypus strictus sister to all other botrychioid species except Helminthostachys. In the ophioglossoid clade, Ophioglossum in the narrow sense was sister to Cheiroglossa plus Ophioderma, but relationships within Ophioglossum were not well supported.