Understanding the spread of Toxoplasma gondii in waterfowl is of interest for elucidating the potential involvement of these birds in maintaining the parasitic life cycle because birds are exposed to these parasites. Sera from 103 adult, free-range game birds, representing 13 different species living in the Italian wetlands, were examined using modified agglutination tests for antibodies specific to T. gondii. In seropositive birds, the brain and heart were homogenized and DNA was extracted to perform nested polymerase chain reaction (nPCR) and genotyping. Out of the 103 serum samples, 9 (8.7%) were scored as positive with titers ranging from 1/20 to 1/160. The seropositive animals corresponded to 4 of the species: Anas platyrhynchos (mallard; 2/17), Anas clypeata (northern shoveller; 2/11), Anas crecca (common teal; 3/41), and Gallinago gallinago (common snipe; 2/8). Parasite DNA was detected in 3 out of 9 brain samples, while the PCR results from the heart specimens were negative in all of the birds. The occurrence of non-clonal types of T. gondii was suggested in all cases. In conclusion, we describe the first documented detection of DNA of T. gondii in tissues from the northern shoveller and common teal, thereby extending the range of intermediate hosts for this parasite.