Increasing numbers of Double-crested Cormorants (Phalacrocorax auritus) in the Mississippi River Delta, USA, have been observed over the past few decades. This piscivorous bird is a definitive host for numerous digenetic trematodes, some of which may cause pathology in a fish host. We conducted a 2-yr survey of intestinal trematodes in 35 Double-crested Cormorants collected in the Mississippi Delta. We counted gastrointestinal trematodes, identified them to species using morphometric and molecular techniques, and sequenced the 18S and cytochrome oxidase I (COI) genes. We collected 4,909 trematodes, representing five digenetic species: Drepanocephalus spathans, Hysteromorpha triloba, Pseudopsilostoma varium, Austrodiplostomum ostrowskiae, and Ascocotyle longa. The most prevalent trematode of the Double-crested Cormorants was D. spathans (91%), followed by H. triloba (78%), P. varium (74%), A. ostrowskiae (57%), and A. longa (29%). Among these, the life cycles are only known for H. triloba and A. longa. Novel DNA sequences of the COI gene were obtained for D. spathans, A. ostrowskiae, P. varium, and A. longa adults. Using these DNA sequences, the identification and confirmation of the larval stages of these parasites in the fish and snail hosts will be possible.