Abstract： Objective To study the microsatellite instability (MSD) of D310 and D16184 located in mitochondrial D-loop region in acute leukemia (AL). Methods The HV-1 and HV-2 regions in D-loop region of 100 persons with the untreated and treated acute leukemia was amplificated and screened by PCR-SSCP,then the abnormal samples was amplificated and sequenced directly and compared with revised Cambridge reference sequence (rCRS) and mtDB. The mutation rates of D310 and D16184 was measured by SPSS11.5 statistics software, x2-test. Results The total mutation rate of D310 was found in 49.0 % (49/100) of our patients. Its mutation rates in untreated group and treated group were 32.5 % (13/40) and 60.0 % (36/60)respectively. The mutation rate of treated group is higher than that in untreated group (P ＜ 0.05). The total mutation rate of D16184 was found in 32.0 % (32/100) of our patients. Its mutation rates in untreated group and treated group were 20.0 % (8/40) and 40.0 % (24/60) respectively. The mutation rate of treated group is higher than that in untreated group (P ＜ 0.05). Conclusion There was a high mutation rate with various types of mutations of microsatellite D310 and D16184 located in mitochondrial D-loop region in AL, which led to a doughty MSI. Chemotherapy could cause a more doughty MSI.