The Wilms' tumor 1 (WT1) antigen is expressed in solid and hematological malignancies， but not healthy tissues， making it a promising target for cancer immunotherapies. Immunodominant WT1 epitopes， the native HLA-A2/WT1 (MFPNAPYL) (HLA-A2/RMFPNAPYL epitope (WT1A)) and its modified variant MFPNAPYL (HLA-A2/YMFPNAPYL epitope (WT1B))， can induce WT1-specific CD8 T cells， although WT1B is more stably bound to HLA-A*02:01. Here， to further determine the benefits of those two targets， we assessed the naive precursor frequencies; immunogenicity and cross-reactivity of CD8 T cells directed toward these two WT1 epitopes. naive WT1A- and WT1B-specific CD8 T cells were detected in healthy HLA-A*02:01 individuals with comparable precursor frequencies (1 in 10-10) to other naive CD8 T-cell pools (for example， A2/HIV-Gag)， but as expected， ~100 × lower than those found in memory populations (influenza， A2/M1; EBV， A2/BMLF1). Importantly， only WT1A-specific naive precursors were detected in HLA-A2.1 mice. To further assess the immunogenicity and recruitment of CD8 T cells responding to WT1A and WT1B， we immunized HLA-A2.1 mice with either peptide. WT1A immunization elicited numerically higher CD8 T-cell responses to the native tumor epitope following re-stimulation， although both regimens produced functionally similar responses toward WT1A via cytokine analysis and CD107a expression. Interestingly， however， WT1B immunization generated cross-reactive CD8 T-cell responses to WT1A and could be further expanded by WT1A peptide revealing two distinct populations of single- and cross-reactive WT1ACD8 T cells with unique T-cell receptor-αβ gene signatures. Therefore， although both epitopes are immunogenic， the clinical benefits of WT1B vaccination remains debatable and perhaps both peptides may have separate clinical benefits as treatment targets.