Tunisia is a highly endemic area for hepatitis A virus (HAV) infection. In the present study, the phylogenetic characterization of the VP1 gene (882 nucleotides) and of the VP1/2A junction (336 nucleotides) of Tunisian strains were examined. One hundred strains isolated from patient with anti-HAV IgM from 2001 to 2004 were amplified by RT-PCR, sequenced at the VP1 and at the VP1/2A junction and aligned with the published sequences to establish phylogenetic analysis. All Tunisian strains belong to genotype I with a greater presence of sub-genotype IA (98%) originate from most of Tunisian regions and 2% of sub-genotype IB. In addition, sub-genotype IA and IB strains formed 25 different clusters. Genetically similar strains were also identified between 2001 and 2004 isolated from the southern and the central part of Tunisia, suggesting that an indigenous strain has been circulating in the Tunisia. The genetic profile of the VP1 region showed that Tun159-02 and Tun40-03 clustered respectively in the IB and IA sub-genotype, however, analysis of VP1/2A junction revealed in contrast that Tun159-02 and Tun40-03 clustered respectively in IA and IB. This is the first report to identify sub-genotype IA in Tunisia and provides new data on the genetic relatedness of HAV from Tunisia and the distribution of sub-genotype IA in this part of the world.