Wild waterfowl and shorebirds in the Delaware-Maryland-Virginia (Delmarva) Peninsula region within the Atlantic Flyway were sampled as part of the Early Detection of Highly Pathogenic H5N1 Avian Influenza (AI) in Wild Migratory Birds program. The U.S. Department of Agriculture (USDA) and state wildlife agencies submitted 7858 samples for AI virus (AIV) testing by real-time reverse transcription PCR (rRT-PCR) to the University of Delaware Poultry Health System from April 2007 to March 2011. Virus isolation attempts were performed on samples with matrix gene cycle threshold (Ct) values ≤33.9. Using rRT-PCR, AIV was detected in 14% (1091/7857) of the samples. In species with sample sizes >100, American black duck (Anas rubripes; 28%), ruddy turnstone (Arenaria interpres; 27%), American green-winged teal (Anas crecca; 21%), semipalmated sandpiper (Calidris pusilla; 27%), greater snow goose (Chen caerulescens atlanticus; 12%), mallard (Anas platyrhynchos; 10%), and northern pintail (Anas acuta; 14%) showed the highest rates of AIV detection. Forty-two AIVs were recovered from eight species: American black duck, mallard, ruddy turnstone, American green-winged teal, greater snow goose, Canada goose (Branta canadensis), ring-necked duck (Aythya collaris), and mallard × American black duck (Anas platyrhynchos × Anas rubripes). Recovered H5 (n = 2) and H7 (n = 2) viruses were found to be low pathogenicity by the USDA National Veterinary Services Laboratory. Additional AIVs represented a diversity of subtype combinations: H1–H4, H6, and H10 and H11 and N subtypes N1–N9 and N6–N9. The rate of AIV recovery from swabbings was inversely related to Ct value, ranging from 50% for Ct values of 16.0–18.9 to 5.1% for Ct values of 31–33.9.