We describe two new species of glassfrogs of the genusCentroleneliving in syntopy at La Enramada, province of Azuay, southwestern Ecuador. They were found in a small creek in montane evergreen forests at 2,900 m elevation. The first new species is distinguished from all other members of the genusCentroleneby having the following combination of characters: dentigerous process of vomer absent; sloping snout in lateral view; thick, white labial stripe and a faint white line between the lip and anterior ¼ of body; humeral spine in adult males; parietal peritoneum covered by iridophores, visceral peritonea translucent (except pericardium); ulnar and tarsal ornamentation; dorsal skin shagreen with dispersed warts; uniform green dorsum with light yellowish green warts; and green bones. The new species is remarkable by being sister to a species from the opposite Andean versant,C. condor. The second new species is distinguished from all otherCentroleneby having the following combination of characters: dentigerous process of vomer absent; round snout in lateral view; thin, yellowish labial stripe with a row of white tubercles between the lip and arm insertion, and a yellowish line between arm insertion and groin; uniform green dorsum; humeral spine in adult males; parietal peritoneum covered by iridophores, visceral peritonea translucent (except pericardium); dorsal skin shagreen with dispersed spicules; ulnar and tarsal ornamentation; and green bones. The second new species is sister toC. sabiniand an undescribed species ofCentrolenefrom southeastern Ecuador. Based on nuclear and mitochondrial DNA sequences, we present a new phylogeny forCentroleneand comment on the phylogenetic relationships inside the genus.