Scinax is a speciose genus of Neotropical hylid frogs. We describe a new species from western Ecuador (elevations between 0 and 1207 m) using morphology, vocalizations, and DNA sequences. We also present a new phylogeny for Scinax based on mitochondrial DNA genes 12S rRNA, Cytochrome Oxidase sub-unit I, Cytochrome B, 16S rRNA, NADH dehydrogenase subunit 1, and adjacent tRNAs. The new species, Scinax tsachila sp. nov. was previously confused with S. quinquefasciatus, a morphologically similar sympatric species. They differ by having markedly different advisement calls, distinct skin texture in the dorsum, and different bone coloration. The new species is sister to S. elaeochroa, a species that differs in advertisement call and color pattern. We provide an updated species account for Scinax quinquefasciatus and a redescription of its holotype.