Influenza A virus (IAV) surveillance in migratory waterfowl in the United States has primarily occurred during late summer and the autumn southern migration. Data concerning the presence and ecology of IAVs in waterfowl during winter and spring seasons in the U.S. northern latitudes have been limited, mainly due to limited access to waterfowl for sampling. The southwestern Lake Erie Basin is an important stopover site for waterfowl during migration periods, and over the past 28 years, 8.72% of waterfowl sampled in this geographic location have been positive for IAV recovery during summer and autumn (June–December). To gain a better understanding of influenza A viral dynamics in waterfowl populations during winter and spring migration (February through April), cloacal swabs were collected from overwintering and spring-migrating waterfowl in Ohio and Michigan in 2006, 2007, 2013, and 2014. A total of 740 cloacal swabs were collected and tested using virus isolation in embryonating chicken eggs, resulting in the recovery of 33 (4.5%) IAV isolates. The influenza A isolates were recovered from eight waterfowl species in the order Anseriformes. Antigenically, the IAV isolates represent 15 distinct hemagglutinin (HA) and neuraminidase (NA) combinations, with seven (21%) of the isolates reported as mixed infections based on antigenic HA subtyping, NA subtyping, or both. This effort demonstrates the presence of antigenically diverse IAV in waterfowl during overwintering and spring migration at northern latitudes in the United States, thereby contributing to the understanding of the maintenance of diversity among waterfowl-origin IAVs.