The Northern Andes harbor one of the highest amphibian diversities worldwide, yet the evolutionary and biogeographic processes shaping this diversity remain insufficiently understood. We investigated the molecular phylogenetics, divergence times, and ancestral area reconstruction of Pristimantis (Craugastoridae) frogs across the Eastern Cordillera of Colombia. Using six gene fragments (12S, 16S, COI, CYTB, RAG1, TYR), we reconstructed a time-calibrated phylogeny for 273 species, representing 41.7% of the genus’ recognized diversity. Our results reveal that Pristimantis species of the Eastern Cordillera do not form a monophyletic group, but instead derive from at least six independent colonization events throughout the Neogene. These colonizations were followed by extensive in situ diversification, tightly associated with phases of accelerated Andean uplift and subsequent climatic shifts. Patterns of divergence suggest that species diversification was shaped by a combination of allopatric isolation, flickering connectivity during Pleistocene climatic oscillations, and parapatric speciation along elevational gradients. Ancestral state reconstructions further indicate that both niche conservatism and ecological divergence contributed to the elevational structure observed across clades. These findings highlight the Eastern Cordillera as a dynamic evolutionary mosaic where geological, climatic, and ecological forces have jointly promoted diversification, offering new insights into the processes driving speciation in tropical montane frogs.
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