Despite the wide distribution and diversity of extant Anguilliformes in tropical America (central western Atlantic and central eastern Pacific), no fossil moray ell (Muraenidae) has been reported to date from this region. Here, we describe the first three fossil specimens of Muraeninae from America: one from the Late Miocene Gatun Formation (Fm.), the second from the Late Pliocene Escudo de Veraguas Fm., both from Panama, and the third from the Late Pliocene Rio Banano Fm. in Costa Rica. Gymnothorax pierreolivieri nov. sp. from the Gatun Fm. is described based on the skull and an in situ otolith. The new species is characterized by a premaxilla-ethmo-vomerine bone (PMx-Etv) with 15 marginal fang-like teeth, two medial teeth, and 13 vomerine teeth arranged in a single row. The dorsal stem of the PMx-Etv forms a very thick and massive structure, with a single anterior foramen located in the anterior tip of the bone. The maxilla has 16 teeth arranged in a double row. The sagitta otolith is elliptic and tapered anteriorly; its posterior margin is smooth and gently rounded; its dorsal margin is slightly arched and elevated posteriorly. The isolated otoliths from the Escudo de Veraguas Fm. in Panama and from the Rio Banano Fm. in Costa Rica are putatively assigned to Echidna sp. and Enchelycore sp., respectively. The diversification of American Muraenidae seems to have been driven by oceanic water interchanges and species dispersal associated to marine currents that flowed eastward through the Central American Seaway prior to the total closure of the Panama Isthmus. The extant American muraenid diversity illustrates allopatric speciation (vicariance) when populations from the Atlantic and the Pacific oceans were isolated by the late Tertiary uplift of the Isthmus of Panama. The palaeoceanographic changes driven by this geological event have had evolutionary consequences on faunal turnover and extinctions through space and time and is reflected by the extant fish diversity.