Abstract Juncaceae are a cosmopolitan family divided into seven genera with a total of about 440 species. The floral anatomy of Juncus and Luzula, the two largest genera, was investigated to provide information for the understanding of the Juncaceae. The anatomical pattern found in both genera is very uniform. The main features shared by the species studied are: tepals with sinuous-walled cells on the abaxial surface and with bilayered margins consisting of upper and lower epidermis; the presence of a supporting tissue in the tepal mesophyll; pollen sac endothecium with spiral thickenings; pollen in tetrads; a furrow in the ovary epidermis facing the dorsal carpellary bundles; and a gynophore. Juncus is distinguished by the presence of stomata on the abaxial epidermis of the tepals, and several ovules with parietal placentation. Luzula differs by the occurrence of phenolic compounds in the tepals; a swelling of the basal region of the tepals and filaments; and a unilocular gynoecium with basal placentation, an ovarian obturator and three ovules. This is the first detailed anatomical description of Juncaceae flowers and their characteristics are useful for delimiting the studied genera and will contribute to the knowledge of the reproductive biology of the family. Swelling of tepal and filament bases is a novel and remarkable peculiarity of Luzula and is probably related to its floral biology, favoring the opening of flowers during anthesis. The presence of an ovarian obturator in Luzula is also first reported here and – besides its taxonomic value – it has a functional importance of guiding pollen tube growth.