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Ovarian tissue cryopreservation can be proposed to patients before gonadotoxic treatments to preserve fertility in case of malignant pathologies like leukemia. Autograft of ovarian tissue is the only available method to restore fertility, but it presents a risk of re-implanting malignant cells. This study aimed to develop a method to detect minimal residual disease in ovarian tissue by multicolor flow cytometry in acute myeloid leukemia. We designed an experimental model consisting in adding acute myeloid leukemia cells to a cell suspension obtained from healthy reference ovarian tissue. Leukemic cell detection within the ovarian cell suspension required the development of a specific myeloid antibody panel different from that commonly used for MRD monitoring in bone marrow. The method was then used to detect minimal residual disease in cryopreserved ovarian tissue from 4 acute myeloid leukemia patients. Multicolor flow cytometry is able to evaluate the presence of viable leukemic cells in the ovarian cortex with good specificity and robust sensitivity of 10-4. We observed a good correlation between multicolor flow cytometry and quantitative polymerase chain reaction results. Ovarian minimal residual disease detection by multicolor flow cytometry was positive in 2 out of 4 acute myeloid leukemia patients. Multicolor flow cytometry can potentially be applied to ovarian tissue from all acute myeloid leukemia patients and is essential to evaluate the risk of cancer re-seeding before autograft of ovarian tissue in case of acute myeloid leukemia.