Chemosymbiosis-based invertebrate communities dominate deep-sea hydrothermal vents and methane seeps, but reliable methods for detecting chemosymbiosis in the fossil record and tracing its evolution into the geologic past are still lacking. Here, we investigate the lipid inventory of shells of four seep-dwelling bivalve species (from both live specimens and empty shells) hosting either methanotrophic or thiotrophic symbionts. All species share a common suite of lipids that are mostly derived from external heterotrophic sources, except for cholesterol and cholestanols in bathymodiolin mussels. The δ13C values of these sterols as low as −83‰ indicate an origin of these compounds from the animal. The transfer was independent of the symbionts being methanotrophic or thiotrophic and thus of the distinct biosynthetic pathways for cholesterol and cholestanol of these animals. This finding opens a new prospect for molecular paleontology by taking advantage of the source specificity and diagenetic stability of these compounds. It could allow tracing chemosymbiosis and its evolution into the fossil record, in particular in bathymodiolin mussels, and potentially also in other metazoan groups.