Abstract We observed a fatal case of Epstein-Barr virus (EBV)-associated hemophagocytic lymphohistiocytosis (HLH) with an abnormal karyotype. Increased levels of αβ T cells of the patient were investigated using an inverse polymerase chain reaction (PCR) of the T-cell receptor variable region gene, followed by Jβ-PCR and single-strand conformation polymorphism (SSCP) to confirm the clonality of specific αβ-T cell subsets. A high frequency (>15%) was recognized in Vβ9 at onset, but not in any Vβ and Vα families 2 weeks after chemotherapy. High levels (>20%) of some Jβ genes were detected in all Vβ families investigated, and the predominant bias of the Jβ2 gene relative to the Jβ1 gene (86.1% versus 13.9% at onset, and 77.4% versus 23.5% after chemotherapy) was recognized in pan-αβ T cells. When each Vβ–Jβ fragment was compared among the samples at onset and after chemotherapy by SSCP analysis, several distinct bands were observed that indicate a clonal evolution. Thus, the findings suggest that some of the αβ T cell clones could be associated with abnormal karyotypes in EBV-HLH. The present findings provide molecular evidence of the presence of oligoclonal T cells in pan-αβ-T cells and clonal evolution during a short clinical course in EBV-HLH with abnormal karyotypes.