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Based on an integrative taxonomical approach, using molecular, morphological, and bioacoustics data, a new species of glassfrog of the genus Centrolene is described from Refugio de Vida Silvestre El Zarza, southern Ecuador. Centrolene zarza sp. nov. is a medium sized species, easily distinguished from all other glassfrogs by its unique combination of characters, such as a shagreen dorsum with elevated warts corresponding to white spots, an evident tympanum, half or more than half of the upper parietal peritoneum covered by iridophores, iridophores absent on all visceral peritonea, including the pericardium, a lobed liver lacking iridophores, males with small projecting humeral spines, the outer edges of forearms and tarsus with a row of enameled warts that often continue into the external edges of Finger IV and/or Toe V, and white or yellowish white iris with thick black reticulations. The new species is closely related to a currently undescribed species and superficially resembles C. condor, C. pipilata, C. solitaria, C. altitudinalis, and C. daidalea. The tadpole and advertisement and courtship calls are described, and the threats to the species survival, mainly represented by habitat loss and contamination due to mining activities, are briefly discussed.