The highlands of the Guiana Shield (Pantepui) in northern South America harbor a unique fauna and flora. However, this diversity remains poorly documented, as many Pantepui massifs remain little explored or unexplored, mainly because their access is very challenging. Considering amphibians, 11 genera are endemic or sub-endemic to Pantepui, and one of them, Neblinaphryne, is monospecific and was recently described from the Neblina massif, at the border between Brazil and Venezuela. We recently undertook an expedition in the nearby, previously uninventoried Imeri massif and discovered a new species of this genus. We describe this new species herein as Neblinaphryne imeri sp. nov., combining molecular, external morphological, acoustic, osteological and myological data. The new species shares with the other Neblinaphryne species (N. mayeri) minuscule septomaxillae and pointed terminal phalanges, confirming the morphological diagnostic characters of the genus. Nevertheless, the new species can promptly be distinguished from N. mayeri by having the head wider than long, a distinct color pattern, and prominent tubercles on the eyelid and humeral region, as well as osteological and genetic differences. These two species are likely endemic to their respective massifs, providing a striking new example of speciation by isolation within Pantepui, which was possibly mediated by climate and elevation, as previously hypothesized for many other lineages endemic to this region.