Billfishes include some of the largest pelagic teleostean species, but several aspects about their morphology, paleobiology, and evolution remain ambiguous. Their fossil record is fragmentary and mostly represented by rostral and skull remains. Here, we present a comparative study of the caudal vertebral morphology of extant istiophorid species and we use this information to describe two fossil vertebrae from the Gatun and Chagres formations, both from the late Miocene of Panama. The caudal vertebra from the Gatun Fm. is characterized by the presence of a lateral apophysis and identified as Makaira sp., while the vertebra from the Río Indio Member of Chagres Fm. lacks this structure and its morphology indicates a different genus. The estimated total length of the Makaira sp. specimen from Gatun Fm. is about 5.18 m, the largest size calculated for a marlin, while the Rio Indio specimen was about 2.56 m long. Phylogenies show that the radiation of most living istiophorid species seemingly occurred during the Pliocene. However, our results highlight that the genus Makaira and the taxa lacking the lateral apophyses occurred during the late Miocene, showing that the radiation of istiophorids most likely occurred before the Pliocene (ca. 5 Ma).