Multiple sclerosis is a chronic inflammatory demyelinating disease of the CNS. Although the aetiology of multiple sclerosis is still unknown, it is widely believed that T cells play a central role in its pathogenesis. To identify and characterize disease‐relevant T cells, we analysed CD4⁺ and CD8⁺ T cells freshly isolated from the CSF and peripheral blood of 36 multiple sclerosis patients for their T‐cell receptor variable β (TCRBV) chain repertoire. In most patients, we found significant overexpression of individual TCRBV chains on CD8⁺ T cells from CSF compared with peripheral blood. In contrast, only a few multiple sclerosis patients showed differences between the two compartments in TCRBV expression on CD4⁺ T cells. The overexpression of specific TCRBV chains on CD8⁺ T cells was found to be stable over several months in selected patients and involved mainly T cells with a memory phenotype. In two patients studied, individual TCRBV chain overexpression was found to be caused by the expansion of T cell populations with identical or highly similar rearranged T‐cell receptor β‐ and α‐chain sequences, which were not found among peripheral blood CD8⁺ T cells. Our findings demonstrate selective enrichment of memory CD8⁺ T cells in the CSF of multiple sclerosis patients, suggesting a role for these CD8⁺ T cells in the pathogenesis of multiple sclerosis. Our study provides a basis for future trials to identify disease‐associated antigens and disease pathogenesis in multiple sclerosis.