The Pliocene Yorktown Formation, deposited on the U.S. Mid-Atlantic Coastal Plain, has played an important role in advancing our knowledge of Pliocene paleoclimate. To refine the age and paleoenvironment of the Yorktown Formation, we analyzed the calcareous nannofossil assemblage and compared it with variations in lithology and calculated sea surface temperature (SST) from previous studies. The Yorktown Formation in the studied sections consists of, in ascending order, the Sunken Meadow, Rushmere, Morgarts Beach, and Moore House members. Sediment samples were collected from these units and analyzed for calcareous nannoplankton assemblages. The last occurrences of both Reticulofenestra pseudoumbilicus (3.82 Ma) and Sphenolithus spp. (3.61 Ma) were recognized within the Sunken Meadow Member. Discoaster tamalis and Discoaster surculus sporadically occurred within the Rushmere Member, but no specimens of the genus Sphenolithus were recorded, suggesting that this unit was deposited sometime between 3.61–2.76Ma. Rare occurrences of the genus Discoaster made it difficult to constrain the age of the Morgarts Beach and Moore House members, but they are most likely deposited before re-entrance of small Gephyrocapsa (ca. 2.5 Ma), supporting previous age estimates based on planktic foraminiferal biostratigraphy and variation in alkenone-based sea-surface temperature estimates. The abrupt decline of both cold-water species (Coccolithus pelagicus) and coastal species (Helicosphaera spp.) is associated with a rise in SST within the Rushmere Member just below the Morgarts Beach Member, and it may reflect a rapid transgression following the global sea-level low stand associated with Marine Isotope Stage (MIS)M2.