We analyzed the occurrence of mitochondrial microsatellite instability (mtMSI) in 262 pairs of female cancer tissues with the matched normal controls. mtMSI was detected in only 4 of 12 microsatellites found in the mitochondrial genome (3 in the D-loop and 1 in the 12S rRNA gene). Interestingly, 95.6% (87/91) of mtMSI was detected in the D-loop, namely, at nucleotide positions 303-315, 514-523, and 16184-16193. This demonstrates that the D-loop is a hotspot for mtMSI. Different incidences of mtMSI at these three microsatellites were found in the four cancer types (including cervical, endometrial, ovarian, and breast). Together with those mtMSI reported in other studies, the differential occurrence of mtMSI at each of the markers in the D-loop region was observed, indicating that the extent of mtMSI varies from one cancer to another. Although the mechanisms of generation and functional impact of mtMSI are still not clear, the high incidence of mtMSI in the D-loop and its broad distribution in human cancers render it a potential marker for cancer detection.