High salinity groundwater more than 1000 metres deep in the Atlantic Coastal Plain of the United States has been documented in several locations1,2, most recently within the 35 million-year-old Chesapeake Bay impact crater3,4,5. Suggestions for the origin of increased salinity in the crater have included evaporite dissolution6, osmosis6, and evaporation from heating7 associated with the bolide impact. Here we present chemical, isotopic and physical evidence that together indicate that groundwater in the Chesapeake crater is remnant Early Cretaceous North Atlantic (ECNA) seawater. We find that the seawater is likely 100-145 million years old and that it has an average salinity of about 70 per mil, which is twice that of modern seawater and consistent with the nearly closed ECNA basin8. Previous evidence for temperature and salinity levels of ancient oceans have been estimated indirectly from geochemical, isotopic and paleontological analyses of solid materials in deep sediment cores. In contrast, our study identifies ancient seawater in situ and provides a direct estimate of its age and salinity. Moreover, we suggest that it is likely that remnants of ECNA seawater persist in deep sediments at many locations along the Atlantic margin.