Despite its negative regulatory role on tumor specific T cells, PD-1 is also a marker of activated tumor-infiltrating T cells. In cancer, PD-1 blockade partially reverses T cell dysfunction allowing the amplification of tumor reactive T cells. Here we investigated the role of PD-1 signaling on effector/memory human T cells specific for shared melanoma antigens, derived from blood. We documented for the first time the existence of melanoma specific T cell clones unable to express PD-1. This stable feature was due to the persistent methylation of the PDCD1 promoter. PD-1pos counterparts, suggesting that high affinity specific T cell clones unable to express PD- 1 are not or rarely present in peripheral blood, as they are probably eliminated by negative selection, due to their high reactivity. We also documented the existence of such PD-1neg T cell clones in melanoma TIL, which also exhibited a lower functional avidity than PD-1pos TIL clones. This clearly shows that PD-1 expression identifies antigen-specific T cell clonotypes of high functional avidity. Finally, we demonstrated that PD-1 blockade during the in vitro selection process of Melan-A specific T cells favored the amplification of higher avidity T cell clonotypes. This preferential amplification of high avidity memory T cells upon PD-1 blockade resonates with the expansion of reactive T cells, including neo-antigen-specific T cells observed in anti-PD-1 treated patients. This feature should also be a useful biomarker of clinical efficiency, while providing new insights for adoptive transfer treatments.