In the present study, epidemiological survey and molecular characterization of hepatitis A virus during an outbreak in five Tunisian childcare centers in El-Mahres during October and November 2006 were carried out. Five well-water and five drinking water samples were included in the present study. Serological investigation and molecular characterization were carried out. All patients were IgM seropositive and the viral genome was detected in all clinical and well-water samples whereas it was not detected in drinking water from the five childcare centers. Sequence analysis showed that all Tunisian strains belong to sub-genotype IA. The genetic profile of the VP1/2A junction showed that the outbreak isolates underwent an amino acid substitution which was absent in virus's strains detected previously in Tunisia. Further studies need to be conducted to evaluate the emergence of the virus's strains in clinical and water samples and more epidemiological data need to be collected about the risk factors which may contribute to acute hepatitis.