T cell epitopes play an important role in the anti-virus specific T cell immunity. Currently, the phenomenon of T cell epitope clustering is reported among different pathogenic microbes. Here, a strategy is established to identify Cytotoxic T Lymphocyte (CTL) epitope clusters region with the combined functional and structural methods. And by using this strategy, a HLA-A2-restricted epitope cluster in SARS-CoV M protein is successfully defined. Firstly, the computer-assisted algorithm was utilized to predict a series of potential HLA-A2 binding peptides around the sequences of two previous identified HLA-A2 epitopes Mn2 and Md3 in the transmembrane domain of SARS-CoV M protein. The HLA-A2 binding affinities of these peptides were accessed through in vitro refolding assay and T2 cell binding assay. Consequently, antigenicity of the peptides was confirmed by ELISPOT by using transgenic mice immunized with peptide Mn2 or DNA vaccine of M protein. Furthermore, the crystal structure of HLA-A^*0201 complexed with peptide Md3-C9 was determined. And the structure shows that the peptide is presented in a traditional conformation of HLA-A2 restricted peptides, however is different from the previously-determined structure of Md3. The result is a great help to understand anti-virus T cell responses and might pave a new way for the T cell-related vaccines for a broader population.