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We report the first records of Scinax ruberoculatus Ferrão, Fraga, Moravec, Kaefer & Lima, 2018 in the state of Amapá, Brazil. We provide an updated distribution map of this species, comment on its occurrence in French Guiana and Suriname, and provide morphometric and bioacoustic data for a population from the municipality of Porto Grande, Amapá, including the first description of the species’ territorial call. This record from Porto Grande extends the distribution of S. ruberoculatus approximately 1430 km northeast from its type locality and helps to better understand its actual distribution.

Previous cytogenetic and phylogenetic analyses showed a high variability in the frog taxa Physalaemus cuvieri and Physalaemus ephippifer and suggested the presence of undescribed diversity in this species complex. Here, by 1) adding specimens from the Brazilian Amazon region, 2) employing sequence-based species delimitation approaches, and 3) including RADseq-style markers, we demonstrate that the diversity in the P. cuvieri–P. ephippifer species complex is even greater than previously suspected. Specimens from Viruá and Western Pará, located at the Guiana Amazonian area of endemism, were recovered as distinct from all previously identified lineages by the phylogenetic analyses based on mitochondrial DNA and RAD markers, a PCA from RAD data, and cytogenetic analysis. The sequence-based species delimitation analyses supported the recognition of one or two undescribed species among these Amazonian specimens and also supported the recognition of at least three other species in the P. cuvieri–P. ephippifer species complex. These new results reinforce the need for a comprehensive taxonomic revision.