Highly pathogenic avian influenza (HPAI) H5N1 has been a global concern since its emergence in 1997, causing widespread outbreaks in birds and sporadic human infections. The clade 2.3.4.4b H5N1 virus has rapidly expanded across continents, infecting numerous mammalian species. In 2024, it was detected in dairy cattle for the first time in the U.S., along with human cases following exposure. In Canada, the first human case of this avian influenza was reported in a critically ill adolescent in late 2024. No human-to-human transmission has been documented, but concerns persist regarding mutations associated with enhanced virulence and human adaptation. Although seasonal influenza vaccines are not directed against H5N1, studies suggest that pre-existing immunity from prior infections or vaccinations may provide partial protection against severe H5N1 infections through cross-reactive immune response. Given the ongoing circulation of avian influenza and the rise in human infections, this study evaluated the effectiveness of neutralizing antibodies developed against seasonal influenza viruses and their cross-reactivity with recent H5N1 strains. Serum samples from 194 retail sector workers in Quebec, collected between late 2021 and 2022, were analyzed using a microneutralization assay. While strong neutralizing activity was found against seasonal influenza viruses, no neutralizing antibodies were detected against H5N1 strains in either vaccinated or unvaccinated individuals. These findings emphasize the need to evaluate cross-reactive antibodies against the neuraminidase protein of H5N1, assess cellular immune responses potentially linked to protection against severe HPAI H5N1 infections and targeted vaccine strategies against recently emerged H5N1 influenza viruses. Keywords: influenza, highly pathogenic avian influenza, H5N1, immunity, cross-reactivity, neutralization.