We collected 6,823 cloacal swabs over four years (2005–2006: 1,460; 2006– 2007: 2,171; 2007–2008: 2,424; and 2008–2009: 768) from 30 potential avian host species. Most samples (88.3 percent) were from dabbling ducks (genus Anas), while diving ducks (genus Aythya) accounted for 5.0 percent, and geese (genera Anser, Chen, and Branta) 3.0 percent of the samples tested. Waterfowl (Anatidae) comprised 98.7 percent of samples, with 1.8 percent from non-migratory dabbling ducks (genus Anas). All samples were screened for avian influenza virus (AIV) by AIV-matrix real-time RT-PCR (rRT-PCR); all rRT-PCR positive samples (541) were processed for virus isolation as well as 4,473 rRT-PCR negative samples. Differences were observed in apparent prevalence estimates over the four years between virus isolation (0.5, 1.3, 3.9, and 0.7 percent) and rRT-PCR (5.9, 6.5, 11.2, and 5.5 percent). We isolated 138 AIVs, of which two were obtained from rRT-PCR negative samples. Unlike previous reports of seasonal variation in AIV prevalence, we documented differences in prevalence estimates among months using rRT-PCR only during 2008–2009 and by virus isolation only during 2006–2007 and 2007–2008. Several of the AIV subtypes we identified are common in North America (e.g., H3, H4, and H6); H3N8 and H4N6 were the most common subtype combinations isolated. Similar to most surveillance studies, we found no significant difference in AIV infection based on host sex, but did find that juveniles were more likely to be positive for AIV than adults. We also documented that dabbling ducks were more likely to be positive for AIV than diving ducks, although not all dabbling ducks are equally likely to be positive. Molecular sequence analysis revealed no insertions of multiple basic amino acids at the cleavage site, which supported the identification of low pathogenic AIV. Phylogenetic anlyses performed on H5, H6, H7, N1, N2, N3, and N4 subtypes sequenced indicated similarity to other North American isolates with the exception of seven H6 which were more similar in amino acid translation to an isolate from Japan. In sum, this is the first multiyear study of avian influenza viruses on waterfowl wintering grounds of the Central Flyway, a historically understudied area of North America.