Four unusual Cenozoic fossil localities are identified here as ancient hydrocarbon seep sites using palaeontological, petrological, and stable carbon isotope data. Late Eocene carbonate-cemented sandstone bodies in the Wagonwheel Mountains in California were previously suspected to represent ancient hydrocarbon seep sites, but the relatively high δ13Ccarbonate values (−12.2 to −5.0‰) suggest oil rather than methane seepage. The Oligocene fauna of the Elmira asphalt mine, Cuba, was previously interpreted as a mix of freshwater and marine taxa, but all species are here identified as belonging to marine groups, including the bivalve families Lucinidae and Vesicomyidae, whose extant members live largely in symbiosis with chemoautotrophic endosymbionts. A carbonate concretion from this site showed δ13Ccarbonate values as low as −32.2‰, which most likely indicates methane seepage. A previously unpublished Oligocene fossil locality in Atlantico, northern Colombia, is dominated by large solemyid, mytilid, lucinid, and vesicomyid bivalves, which most likely lived with chemotrophic endosymbionts. Seepage of biogenic methane without a significant contribution of thermogenic methane is indicated by δ13Ccarbonate values as low as −51.3‰. We confirm that the Pleurophopsis-dominated sites of the Heath Shale and Lomitos Chert in northern Peru are ancient seep sites, although the previous identification of Pleurophopsis peruviana as a vesicomyid is doubtful.