Night frogs (Nyctibatrachidae) form a family endemic to the Western Ghats, a hill chain along the west coast of southern India. Extant members of this family are descendants of a lineage that originated on the subcontinent during its longtime isolation in the Late Cretaceous. Because the evolutionary history of Nyctibatrachidae has always been tightly connected to the subcontinent, these tropically-adapted frogs are an ideal group for studying how patterns of endemism originated and evolved during the Cenozoic in the Western Ghats. We used a combined set of mitochondrial and nuclear DNA fragments to investigate the phylogenetic relationships of 120 ingroup specimens of all known species of Nyctibatrachidae. Our analyses indicate that, although this family had an early origin on the Indian subcontinent, the early diversification of extant nyctibatrachids happened only in the Eocene. Biogeographic analyses show that dispersal across the Palghat gap and Shencottah gap was limited, which led to clade endemism within mountain ranges of the Western Ghats. It is likely that multiple biota have been affected simultaneously by these prominent geographical barriers. Our study therefore further highlights the importance of considering the Western Ghats-Sri Lanka biodiversity hotspot as an assemblage of distinct mountain regions, each containing endemism and deserving attention in future conservation planning.