We studied the molecular basis for CD8 independence of in vivo generated (BM3.3) versus CD8 dependence of in vitro sensitized (KB5.C20/Des) alloreactive H‐2K^b‐specific cytotoxic T lymphocytes (CTL). Using microcapillary high‐performance liquid chromatography fractionation of H‐2K^b eluates, mass spectrometry and CTL reconstitution assays, we determined that BM3.3 and KB5.C20 recognize, respectively, a single peptide (pBM1) expressed on 8,000 H‐2K^b molecules per allogeneic cell, and three distinct peptides (pKB1, 2, 3), each expressed on around 200 H‐2K^b molecules per allogeneic cell. CD8 (in)dependence was intrinsic to the respective TCR/H‐2K^b‐peptide interactions. KB5.C20 and BM3.3 TCR illustrate the correlation that appears to exist between CD8 dependence/low affinity and in vitro sensitization as opposed to low dependency on CD8 and high TCR affinity observed after in vivo sensitization. The results suggest that CD8‐dependent alloreactive CTL obtained in vitro with high frequency correspond to low‐affinity TCR from the MHC‐biased TCR repertoire unpurged by negative selection and have implications for cellular immunotherapeutic approaches.