Niger is a west African country that is highly endemic for hepatitis B virus (HBV) infection. The seroprevalence for HBV surface antigen (HBsAg) is about 20%; however, there are no reports on the molecular epidemiology of HBV strains spreading in Niger. In the present study, HBV isolates from the sera of 58 consecutive, asymptomatic, HBsAg-positive blood donors were characterized. Genotype affiliation was determined by amplification, sequencing and phylogenetic analysis of the preS1, polymerase/reverse transcriptase (RT/Pol) and precore (preC)/C regions. The first series of results revealed that different genomic fragments clustered with different genotypes on phylogenetic trees, suggesting recombination events. Twenty-four complete genomic sequences were obtained by amplification and sequencing of seven overlapping regions covering the whole genome, and were studied by extensive phylogenetic analysis. Among them, 20 (83.3%) were classified unequivocally as genotype E (HBV/E). The remaining four (16.7%) clustered on a distinct branch within HBV/D with strong bootstrap and posterior probability values. Complete molecular characterization of these four strains was achieved by the Simplot program, bootscanning analysis and cloning experiments, and enabled us to identify an HBV/D-E recombinant that formed a new HBV/D subgenotype spreading in Niger, tentatively named D8. Moreover, 20 new complete HBV/E nucleotide sequences were determined that exhibited higher genetic variability than is generally described in Africa. One was found to be a recombinant containing HBV/D sequences in the preS2 and RT/Pol regions. Taken together, these data suggest that, in Niger, genetic variability of HBV strains is still evolving, probably reflecting ancient endemic HBV infection.