Despite continuous virological surveillance (1976–2009) in wild waterfowl (Anseriformes) and shorebirds (Charadriiformes), the ecological and evolutionary dynamics of avian influenza A virus (AIV) in these hosts is poorly understood. Comparative genomic analysis of AIV data revealed that the high prevalence of Charadriiformes infected in Delaware Bay is a reservoir of AIV that is phylogenetically distinct from AIV sampled from most North American Anseriformes. In contrast, influenza viruses sampled from Anseriformes in Alberta are representative of the remaining AIV diversity sampled across North America. While AIV may be restricted to specific migratory flyways over short time frames, our large-scale analysis showed that this population genetic structure was transient and the long-term persistence of AIV was independent of bird flyways. These results suggest an introduced virus lineage may initially be restricted to one flyway, but migration to a major congregation site such as Alberta could occur followed by subsequent spread across flyways. These generalized predictions for virus movement will be critical to assess the associated risk for widespread diffusion and inform surveillance for pandemic preparedness.