The VP60 protein of rabbit hemorrhagic disease virus (RHDV) is a structural protein with important roles in viral replication and assembly. In this study, we immunized BALB/c mice with the RHDV-TP strain. Six monoclonal antibodies (mAbs) were selected and characterized by enzyme-linked immunosorbent assay, Western blotting, and indirectly immunofluorescence analysis (IFA). All six mAbs (AD(4), AG(10), BC9, BE8, BH3, and DE2) had positive reactions with recombinant VP60 as analyzed by IFA, but only two (AG(10) and DE2) reacted with denatured RHDV by Western blotting. Fifty-four partially overlapping fragments of the VP60 gene were expressed with His or Glutathione S-transferase (GST) tags to identify the epitopes recognized by AG(10) and DE2. These two epitopes were located at the C-terminal of VP60 and were longer (64 and 53 amino acids, respectively) than normal B cell epitopes. However, both AG(10) and DE2 also interacted with RHDV2 VP60 expressed in insect cells. Amino acid alignments of the AG(10) and DE2 epitope regions between RHDV and RHDV2 VP60 indicated several mutations, suggesting that the epitopes recognized by the mAbs AG(10) and DE2 were discontinuous. Epitope immunogenicity was evaluated by inoculating specific pathogen-free rabbits with saline, purified DE2 epitope, or RHDV inactive vaccine. Rabbits immunized with the DE2 epitope developed high levels of RHDV-specific antibodies but no cellular immune response and died after challenge with RHDV-HYD isolate. Despite their lack of neutralizing activity, these mAb reagents and epitopes may have useful clinical applications and will be valuable tools in further studies of the structure and function of the RHDV VP60 protein.