In this paper we present an iterative method for delimiting species under the general lineage concept (GLC) based on the multivariate clustering of morphological, ecological, and genetic data. Our rationale is that distinct multivariate groups correspond to evolutionarily independent metapopulation lineages because they reflect the common signal of different secondary defining properties (environmental and genetic distinctiveness, phenotypic diagnosability, etc.) that imply the existence of barriers preventing or limiting gene exchange. We applied this method to study a group of endangered poison frogs, the Oophaga histrionica complex. In our study case, we used next generation targeted amplicon sequencing to obtain a robust genetic dataset that we combined with patterns of morphological and ecological divergence. Our analyses revealed the existence of at least five different species in the histrionica complex (three of them new to science), some of them, occurring in small isolated populations outside any protected areas. The lineage delimitation proposed here has important conservation implications as it revealed that some of the Oophaga species should be considered amongst the most vulnerable of the Neotropical frogs. More broadly, our study exemplifies how multiple -amplicon and multivariate statistical techniques can be integrated to successfully identify species and their boundaries.