Aims Riemerella anatipestifer infections of goslings and ducklings can result in high mortality. Since there are at least 21 serotypes of R. anatipestifer, cross‐protection is an important goal for vaccine development. Methods and Results In this study, we evaluated the immunostimulatory effect of different immunization regimens – the traditional inactivated vaccine vs prime–boost regimens using DNA and protein subunit vaccines (DNA+subunit, subunit+subunit, subunit+inactivated and DNA+DNA). Results showed that, when compared to the inactivated vaccine, prime–boost regimens induced higher and up to 16‐week longer lasting levels of antibody responses, significantly elevated the percentage of the cytotoxic CD8+ T cell and higher expression levels of IFN‐γ, IL‐6 and IL‐12 mRNAs. Furthermore, as an indication of cross‐protection, sera from prime–boost regimens were able to recognize lysates of R. anatipestifer serotypes 1, 2 and 6. Conclusions Prime–boost regimens especially DNA‐prime and protein‐boost, induce strong long‐term immune response and may prove protective for breeder ducks requiring long‐term protection. Significance and Impact of the Study It is worth mentioning that the subunit+inactivated regimen group also elicited strong immune response. The cost of this regimen may only be half of the other prime–boost regimens, making this subunit + inactivated combination an attractive option.