Background Chimeric antigen receptor T-cell (CAR-T) therapy for acute myeloid leukaemia (AML) has thus far been elusive, in part due to target restriction and phenotypic heterogeneity of AML cells. Mutations of the FMS-like tyrosine kinase 3 (FLT3) and DNA methyltransferase 3A (DNMT3A) genes are common driver mutations that present with a poor prognosis in AML patients. We found that AML patients with FLT3 or DNMT3A mutations had higher expression of CD44 isoform 6 (CD44v6) compared to normal specimens. Therefore, we intended to demonstrate CD44v6 could be a specific option for AML with FLT3 or DNMT3A mutations. Methods Internal tandem duplication (ITD) mutations of FLT3 (FLT3/ITD) knock-in clone and DNMT3A-R882H mutant clones of SKM-1 cells were generated using CRISPR/Cas9 and lentiviral transfection, respectively. CD44v6 CAR-T cells were constructed by transfecting T cells with lentivirus containing CD44v6 CAR. CD44v6 expression in AML cell lines, AML patients and healthy donors was evaluated by flow cytometry. DNA methylation assays were used to analyse the mechanisms of FLT3 and DNMT3A mutations affecting CD44v6 expression. Results Aberrant overexpression of CD44v6 was observed in AML cell lines with FLT3 or DNMT3A mutations compared to the wild-type SKM-1 or K562 cells. AML patients with FLT3 or DNMT3A mutations had higher expression of CD44v6 compared to normal specimens. Then we constructed CD44v6 CAR-T cells and found that CD44v6 CAR-T specifically lysed CD44v6(+) cells, accompanied by cytokines release. No significant killing effect was observed from CD44v6(-) AML cells and normal cells after co-culture with CD44v6 CAR-T. These results were also observed in vivo. Furthermore, we found that FLT3 or DNMT3A mutations induced CD44v6 overexpression by downregulating the CpG methylation of CD44 promoter. Conclusions Collectively, CD44v6 is a promising target of CAR-T for AML patients with FLT3 or DNMT3A mutations.