Otoliths are common and diverse in the Neogene of tropical America. Following previous studies of Neogene tropical American otoliths of the lanternfishes (Myctophidae), marine catfishes (Ariidae), croakers (Sciaenidae), and cusk-eels (Ophidiiformes), we describe here the otoliths of the gobies (Gobiidae). The Gobiidae represent the richest marine fish family, with more than 2000 species worldwide and about 250 in America. In the fossil record too they are the species richest family in the Neogene of tropical America. We have investigated otoliths sampled from Ecuador, Pacific and Atlantic Panama, Atlantic Costa Rica, Dominican Republic, Venezuela, and Trinidad, ranging in age from late Early Miocene (late Burdigalian) to late Early Pleistocene (Calabrian). Most of the studied material originates from the collection expeditions of the Panama Paleontology Project (PPP). Our study represents the first comprehensive record of fossil gobies from America, and we recognize 107 species, of which 51 are new to science, 35 are in open nomenclature, and 19 represent species that also live in the region today. Previously, only two fossil otolith-based goby species have been described from the Neogene of tropical America. The dominant gobies in the fossil record of the region are from the Gobiosomatini, particularly of genera living over soft bottoms or in deeper water such as Bollmannia, Microgobius, Antilligobius, and Palatogobius. Another purpose of our study is to provide a first comprehensive account of otoliths of the extant Gobiidae of America, which we consider necessary for an adequate identification and interpretation of the Neogene otoliths. We studied otoliths of 130 extant American gobiid species and figured 106 of them for comparison. We also present a morphological analysis and characterization of the extant otoliths as a basis for the identification of fossil otoliths. Problems that commonly arise with the identification of fossil otoliths and specifically of fossil goby otoliths are addressed and discussed. A comparison of the history of the Gobiidae in tropical America reveals a high percentage of shared species between the Pacific and the Atlantic basins during the Late Miocene (Tortonian and Messinian) from at least 11 to 6 Ma. A recording gap on the Pacific side across the Pliocene allows a comparison again only in the late Early Pleistocene (Calabrian, 1.8 to 0.78 Ma), which shows a complete lack of shared species. These observations support the effective closure of the former Central American Seaway and emersion of the Isthmus of Panama in the intervening time. Groups that today only exist in the East Pacific were also identified in the Miocene and Pliocene of the West Atlantic, and there is also at least one instance of a genus now restricted to the West Atlantic having occurred in the East Pacific as late as the Pleistocene. The evolution of gobies in tropical America and the implications thereof are extensively discussed. Furthermore, observations of fossil gobies in the region are discussed in respect to paleoenvironmental indications and paleobiogeographic aspects.