Abstract Background Millions of people worldwide are infected with Hepatitis A Virus (HAV) annually - the leading widely spread causes of acute viral hepatitis in developing countries across all continents. Objective To evaluate the prevalence of anti-HAV antibodies; to determine HAV genotypes by nucleotide sequencing in patients suspected of acute hepatitis at public healthcare institutions; and to assess the association between this infection with age, gender, region, and other risk factors Methods Between October 2019 and May 2023, we analyzed 1,562 patients with more than 18 years old with clinical suspected acute hepatitis. Results Out of the 1,562 individuals, 1,244 were seropositive for Anti-HAV IgG, a median age of 45 years (IQR: 35 to 56) vs. 31 years (IQR: 25 to 39) in non-reactive individuals (p<0.001). A higher seropositivity was found in men compared to women (p=0.002). North region showed the highest seropositivity (p<0.001). There was also an association between poor sanitation conditions and seropositivity, such as lack of sewage collection and consumption of untreated water (p<0.001), as well as an association between heterosexual orientation and higher likelihood of seropositivity (p=0.015). In the logistic regression analysis for anti-HAV IgG seropositivity, age and male sex were significantly associated with seropositivity (OR = 1.09, 95% CI: 1.08 to 1.11, p<0.001; OR = 1.56, 95% CI: 1.16 to 2.11, p=0.004). North region showed a significant increase in the odds for seropositivity compared to all other regions. For anti-HAV IgM, out of the 1,562 individuals, 97 were seropositive, significantly higher in younger individuals [median age of 34 years (IQR: 28 to 40) vs. 42 years (IQR: 32 to 54) in non-reactive individuals (p<0.001)] and in males (p<0.001). South and Southeast regions had higher seropositivity (p<0.001). Non-heterosexual orientation and more than 2 sexual partners significantly correlated with seropositivity (p<0.001). In the logistic regression analysis for anti-HAV IgM, age showed an inverse association with seropositivity (OR = 0.96, 95% CI: 0.94 to 0.98, p<0.001), being more frequent in men (OR = 1.86, 95% CI: 1.11 to 3.21, p=0.021). Consumption of seafood was significantly associated with increased odds of seropositivity for anti-HAV IgM (OR = 2.26, 95% CI: 1.43 to 3.56, p<0.001). South and Southeast regions showed more IgM positive cases than North, Northeast and Midwest regions. Among the 1,562 cases, 35 complete (or almost complete) HAV genomes were successfully recovered from metagenomics sequencing data. These genomes range from 6,625 to 7,462 bp in length (89-100% completeness), sequences were fairly similar in pairwise nucleotide alignment, classified as genotype I.A, and displaying identities ranging from 94.7 to 99.7%. Conclusions Hepatitis A infection was found among 6.2% acute hepatitis cases attended in public services in Brazil while previous infections or vaccinated patients were found in 78.9% patients. While anti-HAV IgG positive patients where most frequent in the North region of Brazil, acute hepatitis A (IgM-positive) were more frequent in South and Southeast regions. Our results reinforce the need to implement vaccination against hepatitis A among individuals with more than 18 years in our country.