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The Príncipe giant tree frog Leptopelis palmatus is endemic to the small oceanic island of Príncipe in the Gulf of Guinea. For several decades, this charismatic but poorly known species was confused with another large tree frog species from continental Africa, L. rufus. Phylogenetic relationships within the African genus Leptopelis are poorly understood and consequently the evolutionary history of L. palmatus and its affinity to L. rufus remain unclear. In this study, we combined mitochondrial DNA (mtDNA), morphological, and acoustic data for L. palmatus and L. rufus to assess different axes of divergence between the species. Our mtDNA gene tree for the genus Leptopelis indicated that L. palmatus is not closely related to L. rufus or other large species of Leptopelis. Additionally, we found low mtDNA diversity in L. palmatus across its range on Príncipe. We found significant morphological differences between females of L. rufus and L. palmatus, but not between males. We characterised the advertisement call of L. palmatus for the first time, which is markedly distinct from L. rufus. Finally, we summarised our observations of L. palmatus habitats and additional notes on phenotypic variation and behaviour. Our study reinforces the distinctiveness of L. palmatus and provides information important for the conservation of this endangered species. [ABSTRACT FROM AUTHOR]