Paralichthys lethostigma (Southern Flounder) inhabits the continental shelf and estuarine waters of the Gulf of Mexico and the east coast of the North Atlantic, from peninsular Florida to Albemarle Sound in North Carolina. Between 30 May and 20 August 2012, we collected 15 juvenile (71–192 mm) Southern Flounder in fyke nets in the Mattaponi River, a tributary of the York River, in southeastern Virginia. This is the first known documentation of juvenile Southern Flounder in any tributary of Chesapeake Bay. We confirmed our identification of the specimens as Southern Flounder morphologically and genetically by counting gill rakers and sequencing cytochrome oxidase subunit I, respectively.