Duck hepatitis is a convenient model of hepatitis B virus (HBV) infection, but the lack of immunological reagents hampers investigation of pathogenesis and vaccine development. The aim of this study was to define T-cell epitopes in the surface peptide recognized by vaccinated immune birds. Blastogenesis assays were used to test the proliferative response of spleen mononuclear cells to synthetic peptides spanning the pre-S/S region in 22 naïve and 13 immunized and challenged immune ducks. Roughly ≥50% of the immune ducks responded to five immunodominant peptides eliciting a statistically greater proliferative response than in naïve birds. Fewer ducks responded to an additional six peptides. No statistically significant difference could be shown for the response to 11 peptides between the immune ducks and the naïve ducks. There was no clustering of the immunodominant peptides which were located throughout the surface antigen at sites of major swings in hydrophobicity. A number of peptides which induce lymphoblastogenesis in vaccinated immune ducks have been identified. Their role in spontaneous recovery from duck hepatitis B infection merits investigation.