Abstract Allogeneic hematopoietic cell transplantation (HCT) has a well-documented ability to cure a number of malignant hematological diseases. The curative principle in allogeneic HCT is the Graft-versus-Leukemia (GVL) effect and nonmyeloablative (NMA) conditioning HCT relies exclusively on this anti-tumor effect to eliminate tumor cells. Donor T-cells are documented to be responsible for the GVL effect, however, often they also cause Graft-versus-Host disease (GVHD) which is associated with high morbidity and mortality. Characterization of cells and molecules involved in both GVL and GVHD would potentially set the stage for separation of GVL and GVHD in order to augment GVL in the absence of GVHD. In the present study, we analyzed the clonotype composition of CD8+ T cells following NMA conditioning and HCT, in two patients with chronic lymphocytic leukemia (CLL). T-cell receptor (TCR) clonotype mapping (RT-PCR combined with denaturing gradient gel electrophoresis (DGGE)) was used to identify clonally expanded CD8+ T cells in blood samples. This method provides a “molecular fingerprint” of each unique T cell based on junctional diversity of the TCR CDR3 region and, thus, offers the means to track T-cell clonotypes in time and space. Longitudinal comparative analyses showed that CD8+ T-cell clonality was highly dynamic during early phases after transplantation with various clonotypes emerging and disappearing. However, clonal diversity decreased after 4–5 months and stable CD8+ T-cell clonotypes appeared and persisted throughout the analyzed period (up to two years). One patient received donor lymphocyte infusion (DLI) due to disease progression and this was shown to lead to establishment of recurrent (detected prior to DLI) CD8+ T-cell clonotypes as well as new CD8+ T-cell clonotypes. The appearance of these cells correlated with disease remission strongly suggesting their engagement in anti-CLL reactivity. To examine the functional capacities of clonally expanded T cells after HCT, recipient CD8+ T cells were stimulated ex vivo with pre-transplant patient CLL cells and/or normal hematopoietic cells and the T-cell surface expression of CD107a (marker for cytotoxicity) was detected by FACS. Clonotype mapping analyses of FACS sorted CD107a positive CD8+ T cells after stimulation with CLL cells demonstrated that such cytotoxic CD8+ T cells were present as stable clonally expanded T cells in vivo strongly implying their involvement in an ongoing anti-CLL-response. Furthermore, co-culture with normal hematopoietic cells resulted in a unique CD107a positive expanded T-cell clonotype. Our results strongly suggest that clonally expanded CD8+ T cells are involved in an ongoing tumor response and support data which demonstrate that GVL and GVHD are the result of distinct responses.