Uranium-lead geochronology in detrital zircons and provenance analyses in eight boreholes and two surface stratigraphic sections in the northern Andes provide insight into the time of closure of the Central American Seaway. The timing of this closure has been correlated with Plio-Pleistocene global oceanographic, atmospheric, and biotic events. We found that a uniquely Panamanian Eocene detrital zircon fingerprint is pronounced in middle Miocene fluvial and shallow marine strata cropping out in the northern Andes but is absent in underlying lower Miocene and Oligocene strata. We contend that this fingerprint demonstrates a fluvial connection, and therefore the absence of an intervening seaway, between the Panama arc and South America in middle Miocene times; the Central American Seaway had vanished by that time.