A new species of Niceforonia is described from the montane cloud forests of southwestern Colombia. The new species is distinguished from all congeners by possessing dorsolateral folds, a visible tympanic membrane, a fleshy lip swelling in adult males, nuptial pads, circumferential grooves on all fingers, a reddish-brown to grayish dorsal coloration, yellow ventral and hidden surfaces, and dark supra-inguinal spots. Molecularly, the new species differs from all congeners by an uncorrected p-distance of > 7% for the 16S rRNA gene fragment. Furthermore, the possession of dark supra-inguinal spots optimizes unambiguously as a synapomorphy for two clades within Strabomantidae. The first clade comprises members of the genus Niceforonia and includes N. brunnea, N. dracula, N. elassodiscus, N. mantipus, N. nigrovittata, N. quilloturo, N. peraccai, four candidate species, and the new species described herein. This clade is referred to as the Niceforonia nigrovittata species group, diagnosed by the presence of dark supra-inguinal spots. By retaining this name, we ensure taxonomic stability and honor the well-established historical use of the nomenclature in literature. The second clade comprises members of the subfamily Holoadeninae, including the genera Bahius, Barycholos, Phyllonastes, and Urkuphryne. A previous study already proposed a phenotypic synapomorphy for the group composed of Phyllonastes + Barycholos. Consequently, the available evidence corroborates the monophyly of both the group comprising these four holoadenine genera and the Phyllonastes + Barycholos group. This congruence provides a robust, nested phylogenetic resolution and precise taxonomic diagnoses, demonstrating how the integration of molecular and phenotypic evidence is a powerful tool for testing evolutionary hypotheses in brachycephaloid frogs.