AbstractHighly pathogenic avian influenza (HPAI) H5N1 clade 2.3.4.4b viruses were first detected in St. John’s, Canada in late 2021. To investigate the patterns of avian influenza virus (AIV) infection and immune responses subsequent to the arrival of H5N1, we sampled the wild urban duck population in this area for a period of 16 months after the start of the outbreak and compared these findings to those from archived samples. Antibody seroprevalence was relatively stable before the outbreak (2011–2014) at 27.6% and 3.9% for anti-AIV (i.e., NP) and H5-specific antibodies, respectively. During the winter of 2022, AIV-NP and H5-specific antibody seroprevalence both reached 100%, signifying a population-wide infection event, which was observed again in late February 2023 following a second H5N1 incursion from Eurasia. As expected, population-level immunity waned over time, with ducks seropositive for anti-AIV-NP antibodies for approximately twice as long as for H5-specific antibodies, with the population seronegative to the latter after approximately six months. We observed a clear relationship of increasing antibody levels with decreasing viral RNA loads that allowed for interpretation of the course of infection and immune response in infected individuals and applied these findings to two cases of resampled ducks to infer infection history. Our study highlights the value of applying both AIV surveillance and seroprevalence monitoring to provide a better understanding of AIV dynamics in wild populations, which may be crucial following the global dissemination of clade 2.3.4.4b H5Nx subtypes to assess the threats they pose to both wild and domestic animals, and to humans.