The Wilms’ Tumour 1 (WT1) antigen is expressed in solid and haematological malignancies, but not healthy tissues, making it a promising target for cancer immunotherapies. Immunodominant WT1 epitopes, the native HLA-A2/WT1126-134 (RMFPNAPYL) (WT1A) and its modified variant YMFPNAPYL (WT1B), can induce WT1-specific CD8+ T-cells, although the modified variant WT1B epitope appears to be more stable at HLA-A*02:01 binding. Here, to further determine the benefits of those two targets, we assessed the naïve precursor frequencies; immunogenicity and cross-reactivity of CD8+ T-cells directed towards these two WT1 epitopes. Ex vivo naïve WT1A- and WT1B-specific CD8+ T cells were detected in healthy HLA-A*02:01+ individuals with comparable precursor frequencies (1 in 105-106) to other naïve CD8+ T cell pools (e.g. A2/HIV-Gag77-85), but expectantly ~100× lower than those found in memory populations (influenza, A2/M158-66; EBV, A2/BMLF1280-288). Interestingly, only WT1A-specific naïve precursors were detected in HLA-A2.1 transgenic mice. To further assess the immunogenicity and recruitment of CD8+ T cells responding to WT1A and WT1B, we immunized HLA-A2-expressing transgenic mice with either peptide. WT1A-immunization elicited numerically higher CD8+ T-cell responses to the native tumour epitope following in vitro re-stimulation, although both regimens produced functionally similar responses towards WT1A. Interestingly however, WT1B-immunization generated cross-reactive CD8+ T-cell responses to WT1A and could be further expanded by the WT1A peptide revealing two distinct populations of single- and cross-reactive WT1A+CD8+ T cells with unique TCRab gene signatures. Therefore, while both epitopes are immunogenic, the clinical benefits of WT1B vaccination remains debatable and perhaps both peptides may have clinical benefits as treatment targets depending on the situations.