Despite significant improvement in our understanding of T-cell acute lymphoblastic leukemia (T-ALL) biology and pathogenesis, many questions remain unanswered. In previous studies, we found a T-ALL case with two malignant T-cell clones with Vδ1Dδ2Dδ3Jδ1 and Vδ2Dδ3Jδ2 rearrangements. In this study, we further characterized T-ALL cases with two malignant clones containing Vδ1Dδ3Jδ1 and Vδ2Dδ1Jδ1 rearrangements using fine-tiling array comparative genomic hybridization, ligation-mediated polymerase chain reaction (LM-PCR), sequencing, and reverse transcription polymerase chain reaction (RT-PCR) analysis. We further analyzed the distribution and clonality of the T-cell receptor (TCR) Vγ and Vδ subfamily T cells in the two T-ALL cases by RT-PCR and GeneScan. Monoclonal Vδ1 and Vδ2 subfamilies were confirmed in both samples, the Vδ3 through Vδ7 subfamilies could not be detected in the T-ALL samples, whereas the oligoclonal Vδ8 subfamily could be identified. Based on the clinical finding that both of the T-ALL cases with two malignant T-cell clones had a poor outcome, we attempted to compare the expression pattern of genes related to T-cell activation and proliferation between cases with the malignant Vδ1 and Vδ2 T-cell clones and T-ALL cases with a mono-malignant Vα T-cell clone. We selected two T-ALL cases with VαJα rearrangements and analyzed the expression level of Notch1, TAL1, and the CARMA-BCL10-MALT-A20-NF-κB pathway genes by real-time PCR. A20 had significantly higher expression in the biclonal compared with the monoclonal T-ALL group (p=0.0354), and there was a trend toward higher expression for the other genes in the biclonal group with the exception of TAL1, although the differences were not statistically significant. In conclusion, we identified two T-ALL cases with biclonal malignant T-cell clones and described the characteristics of the biclonal T-ALL subtype and its gene expression pattern. Thus, our findings may improve the understanding of biclonal T-ALL.