Display omitted ► Taxonomic groupings within the frog genus Pristimantis are unsupported by our 5-gene phylogenetic analysis. ► Within the genus Pristimantis we estimate at least 11 independent colonization events from South to Central America. ► At least eight invasions took place before 3.5million years ago, the assumed date for the completion of the Isthmus. ► Land connections between Central and South America may have formed earlier than is often assumed. ► The old age of lineages endemic to Panama suggests the presence of cryptic taxa. The completion of the land bridge between North and South America approximately 3.5–3.1million years ago (Ma) initiated a tremendous biogeographic event called the Great American Biotic Interchange (GABI), described principally from the mammalian fossil record. The history of biotic interchange between continents for taxonomic groups with poor fossil records, however, is not well understood. Molecular and fossil data suggest that a number of plant and animal lineages crossed the Isthmus of Panama well before 3.5Ma, leading biologists to speculate about trans-oceanic dispersal mechanisms. Here we present a molecular phylogenetic analysis of the frog genus Pristimantis based on 189 individuals of 137 species, including 71 individuals of 31 species from Panama and Colombia. DNA sequence data were obtained from three mitochondrial (COI, 12S, 16S) and two nuclear (RAG-1 and Tyr) genes, for a total of 4074 base pairs. The resulting phylogenetic hypothesis showed statistically significant conflict with most recognized taxonomic groups within Pristimantis, supporting only the rubicundus Species Series, and the Pristimantis myersi and Pristimantis pardalis Species Groups as monophyletic. Inference of ancestral areas based on a likelihood model of geographic range evolution via dispersal, local extinction, and cladogenesis (DEC) suggested that the colonization of Central America by South American Pristimantis involved at least 11 independent events. Relaxed-clock analyses of divergence times suggested that at least eight of these invasions into Central America took place prior to 4Ma, mainly in the Miocene. These findings contribute to a growing list of molecular-based biogeographic studies presenting apparent temporal conflicts with the traditional GABI model.