Water and bed-sediment samples were collected from selected water bodies along the Atlantic Flyway and analyzed for the presence of viable avian influenza viruses. Samples were collected during February and May 2006 and January 2007 at U.S. Fish and Wildlife Service National Wildlife Refuges in Georgia, South Carolina, North Carolina, Virginia, and Maryland. Avian influenza viruses were detected in samples collected from the Savannah National Wildlife Refuge in Georgia during February 2006 and from the Santee National Wildlife Refuge in South Carolina and the Pee Dee National Wildlife Refuge in North Carolina during January 2007. Avian influenza virus was detected in water temperatures ranging from 11.8 to 12.7 degrees Celsius when birds were either present or had departed at least 10 days prior to sampling. Although the literature indicates that avian influenza virus persists in the environment more effectively at colder temperature regimes, these detections were made in a comparatively warmer climate at a time of the year when cooler water temperatures prevail.