Multiparameter flow cytometry (MFC) identifies rare cases of biclonal disease in chronic lymphocytic leukaemia (CLL). By MFC, we identified 76 patients with biclonal disease in a cohort of 5523 CLL patients (1·4%). Fluorescence in situ hybridization and chromosome banding analysis revealed five and six cases, respectively, with two different cytogenetic aberrations due to clonal evolution. Two different B-cell receptor rearrangements and IGHV subtypes were more frequent in biclonal than in monoclonal CLL by MFC (37·1% vs. 2·7%; P < 0·001). Patients with biclonal CLL by MFC showed a trend to a shorter time to treatment than monoclonal CLL (P = 0·080).