Doryopteris triphylla (Pteridaceae-Cheilanthoideae) grows in xeric habitats in Brazil, Paraguay, Uruguay and Argentina. The aim of this study was to characterize D. triphylla anatomically, histochemically and cytogenetically. For anatomical characterization, rhizomes, roots, petioles and leaves were made and then stained using Safranine-Astra Blue for further observations. Leaf blades were also cleared. For histochemical analysis, leaf cross sections were stained with different reagents to identify glandular trichomes compounds. For cytogenetic characterization, a karyogram was performed using laboratory cultivated roots. Results show a dictyostelic rhizome covered with scales with apical secreting gland; diarch roots; petiole cross-sections show thick cuticle, uniseriate epidermis, parenchymatic cortex cells with thick walls and a vascular bundle with two xylem groups; and hypostomatic fronds with glandular trichomes. Histochemical studies of secretion products of the glandular trichomes were positive for polysaccharides, pectins, lipids, acid lipids, dihydroxyphenols, phenols and flavonoids. Cytogenetically, D. triphylla is described as a diploid species (2n = 60), with chromosomes gradually decreasing in size. The apical glands in scales of rhizomes, the presence of two xylem groups in the vascular bundle in the petiole and the glandular trichomes on the abaxial surface are new contributions to the species. The type of chemical products secreted by glandular leaf trichomes and karyotype estimation is shown for the first time in this species.