Adoptive cell transfer between genetically identical hosts relies on the use of a congenic marker to distinguish the donor cells from the host cells. CD45, a glycoprotein expressed by all hematopoietic cells, is one of the main congenic markers used because its two isoforms, CD45.1 and CD45.2, can be discriminated by flow cytometry. As a consequence, C57BL/6J (B6; CD45.2) and B6.SJL-Ptprc^a Pepc^b/BoyJ (B6.SJL; CD45.1) mice are widely used in adoptive cell transfer experiments, under the presumption that they differ only at the CD45 (Ptprc) locus. However, recent studies have identified genetic variations between these congenic strains and have notably highlighted a differential expression of cathepsin E (CTSE). The B6.SJL mouse presents a number of functional differences in hematopoietic stem cell engraftment potential and immune cell numbers compared with the B6 mouse. In this study, we showed that B6 and B6.SJL mice also differ in their CD8⁺ T cell compartment and CD8⁺ T cell responses to viral infection. We identified Ctse as the most differentially expressed gene between CD8⁺ T cells of B6 and B6.SJL and demonstrated that the differences reported between these two mouse strains are not due to CTSE. Finally, using CRISPR–Cas9 genome editing, we generated a CD45.1-expressing B6 mouse by inserting one nucleotide mutation (A904G) leading to an amino acid change (K302E) in the Ptprc gene of the B6 mouse. We showed that this new B6-Ptprc^{em(K302E)Jmar}/J mouse resolves the experimental biases reported between the B6 and B6.SJL mouse lines and should thus represent the new gold standard for adoptive cell transfer experiments in B6.