BackgroundThe Tropical Andes is the world’s most biodiverse hotspot. This region contains >1,000 amphibian species, more than half of which are endemic. Herein we describe two new glassfrog species (Centrolenidae:Hyalinobatrachium) that we discovered within relatively unexplored and isolated localities of the Ecuadorian Andes.MethodsWe employed morphological, acoustic, and molecular methods to test the hypothesis thatHyalinobatrachium mashpisp. nov andH. nounssp. nov. are species new to science. Following standard methods, we generated mitochondrial sequences (16S) of 37 individuals in the genusHyalinobatrachium. We inferred the phylogenetic relationships of the two new species in comparison to all other glassfrogs using Maximum Likelihood. In addition to describing the call ofH. mashpisp. nov., we performed a discriminant analysis of principal components (DAPC) with the advertisement call characteristics of several congeners.ResultsBased on an integrative taxonomy approach, we describe two new species. Morphological traits and the inferred phylogeny unambiguously place the new taxa in the genusHyalinobatrachium. Both species are distinguished from other glassfrogs mainly by their dorsal coloration (i.e., dorsum lime green with small light yellow spots, head usually with interorbital bar) and transparent pericardium (i.e., the heart is visible through the ventral skin). The new species exhibit a high morphological similarity (i.e., cryptic) and occur within relatively close geographical proximity (closest aerial distance = 18.9 km); however, their uncorrectedpdistance for the mitochondrial gene 16S is 4.6–4.7%, a value that greatly exceeds the genetic distance between closely related species of centrolenid frogs. The DAPC revealed that the advertisement call ofH. mashpisp. nov. is acoustically distinct.DiscussionOur findings are congruent with several previous studies that report a high degree of endemism in the Toisán mountain range, which appears to be isolated from the main Andean cordillera for some amphibian groups. We recommend that bothH. mashpisp. nov. andH. nounssp. nov. be listed as Endangered, following IUCN criteria. These new species provide another example of cryptic diversity in the Andes—further evidence that the region fosters much more biodiversity than we have the resources to catalog. Threatened by mining and other exploitative industries, these glassfrogs and many other yet-to-be-discovered Andean species highlight the dire need for effective conservation measures—especially in northwestern Ecuador.