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Objective: To evaluate the significance of two-color interphase fluorescence in situ hybridization (FISH) using X and Y centromere probe in the engraftment estimation and minimal residual disease (MRD) monitoring after allogeneic stem cell transplantation (alloSCT). Methods: Samples from 12 cases patients in different periods after alloSCT were detected by interphase FISH. Results: All of the 12 patients were proved to obtain engraftment 22(35 days after alloSCT. While traditional karyotype showed as 100%XX or 100%XY invariably, FISH showed different percentages of donor original sex chromosome. Conclusion: Two-color interphase FISH is a more sensitive and simple test for engraftment evaluation and MRD monitoring post SCT, though, it can not entirely replace traditional karyotype analysis and gene detection by RT-PCR.