We present the statistical analysis of diversifying selective pressures on the hepatitis D antigen gene (HDAg). Thirty-three distinct HDAg sequences from subtypes I, II, and III were tested for positive selection using maximum likelihood methods based on models of codon substitution that allow variable selective pressures across sites. Such methods have been shown to be sufficiently accurate and suc- cessful in detecting positive selection in a variety of viral and nonviral protein-coding genes. About 11% of codon sites in HDAg were estimated to be under diversifying selection. Remarkably, most of the resi- dues predicted to evolve under positive selection were located in the immunogenic domain and the N-ter- minusregionwithreportedantigenicactivity.These sites are potential targets of the host’s immune re- sponse. Identification of residues mutating to escape immune recognition may help to distinguish the most virulent strains and aid vaccine design. Possible interplay between positive selection and recombination on the gene is discussed but no significant evi- dence for recombination was found.