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Background: Until 2005, when a single dose of vaccine was implemented in one-year-old children, the Hepatitis A virus (HAV) was responsible for approximately 90% of acute hepatitis cases in the paediatric population in Argentina. However, despite vaccination success, sporadic outbreaks of HAV still occur among adults. This study aimed to assess the seroepidemiology of HAV in Argentina, analysing IgG and IgM antibodies against HAV in a large population, both vaccinated and unvaccinated. Methods: The study included 16,982 patients attending a hospital from 2001 to 2023. The cohort was divided into two groups: 16,638 individuals who were not reached by the vaccination program implemented in 2005 and 344 children who were covered by the universal vaccination. Results: Anti-HAV IgG was detected in 56.7% of cases. The rate was significantly higher in individuals born after 2005 (77.7%) compared to those born before (56.3%), p < 0.001. The age groups 19–40 and 41–60 years showed the anti-HAV IgG lowest rates. On the other hand, 100/3956 cases (2.5%) with suspected acute hepatitis were positive for Anti-HAVIgM. Notably, none of these were born after the mandatory vaccine rollout. Conclusions: The study of this large cohort contributes to the understanding of the seroepidemiology of HAV. Although the implementation of the vaccine achieved its main goal, the age segment between 19 and 60 years does not reach the estimated threshold to achieve herd immunity. These findings reveal the importance of targeting vaccination campaigns, provide essential insights for public health planning, and guide future immunisation strategies against HAV in Argentina. [ABSTRACT FROM AUTHOR]