The root, rhizome, petiole and blade anatomy of Ctenitis melanosticta was studied and compared with the available information for closely related genera. Root is diarc with sclerenchyma and parenchyma cells in the cortex, with fungal hyphae exclusively in the latter. The occurrence of sclereid nests in the rhizome is shared with Dryopteris and Campyloneurum. A cortical band was present in petiole and lamina, as in Dryopteris and other genera of the most derived ferns. The lamina had unifacial mesophyll and under the adaxial epidermis there were several layers of the fiber, as described for Elaphoglossum and Thelypteris, but differed from Asplenium, Dryopteris, and Polybotrya with collenchyma. The anatomy of C. melanosticta was similar to that of most species studied of Dryopteridaceae, although with some differences. Additional anatomical studies in species of Dryopteridaceae will allow to confirm the diagnostic value of several anatomical features, such as the lack of cortical band in the rhizome, the sclereid nests, the unifacial mesophyll, and the cortical band in the lamina.