Flow cytometric T-cell receptor (TCR)-V(β) repertoire analysis (TCR-V(β)-R) is a sensitive method for detection of T-cell clonality; however, no uniform approach exists to define clonality in neoplastic T cells. TCR-V(β)-R was evaluated in patients with a diagnosis of T-cell neoplasia in initial diagnostic specimens from 41 patients and for minimal residual disease (MRD) monitoring in 61 sequential samples from 14 patients with mature T-cell neoplasia. Gating strategies and criteria for detection of T-cell clonality were determined. In all 41 initial specimens, T-cell clonality was demonstrated via TCR-V(β)-R. The frequency of V(β) usage was consistent with random neoplastic transformation of TCR-V(β) subsets. MRD was successfully detected in follow-up samples from all 14 patients evaluated, Furthermore, MRD after therapy was quantitated in 48 peripheral blood specimens. TCR-V(β)-R analysis is a sensitive method for detection of T-cell clonality and is useful for diagnosis and MRD detection in multiple specimen types.