The high molecular weight melanoma-associated antigen (HMW-MAA) is highly expressed in advanced primary and metastatic melanoma. An epitope of the core protein of HMW-MAA is recognized by the murine monoclonal antibody (mAb) 225.28S. In this study, we aimed to characterize peptides that antigenically mimicked this epitope and to determine their efficacy as components of an HMW-MAA-based anti-melanoma vaccine. Therefore, we screened a constrained 10 mer phage display peptide library against mAb 225.28S. Selected phage-displayed peptides were then tested for their specificity for the antibody's antigen-binding site. DNA sequences coding for specific peptide ligands were determined. Binding of mAb 225.28S to HMW-MAA was inhibited in a dose-dependent manner by phage-displayed peptides from 51 to 83% and by synthetic peptides from 38 to 87%. Subsequently, the immunogenicity of the five mimotopes with the highest inhibition capacity was examined in rabbits. Immunizations with synthetic mimotopes conjugated to tetanus toxoid resulted in peptide-specific antibodies, but none of the highly antigenic mimotopes induced HMW-MAA cross-reactive antibodies. This report describes an example of disparity between antigenicity and cross-reactive immunogenicity, complicating the selection of potential vaccine candidates.