Mitochondrial DNA (mtDNA) is known for its high frequencies of polymorphisms and mutations. The non-coding displacement (D)-loop, especially a mononucleotide repeat (poly-C) between 303 and 315 nucleotides (D310), has been recently identified as a frequent hotspot of mutations in human neoplasia, including breast cancer. To further explore the sequence variations of mitochondrial D-loop region in familial breast cancer and their possible associations with breast cancer risk, PCR-SSCP and direct DNA sequencing methods were used to detect the variants of the mtDNA D-Loop in 23 familial breast cancer patients as well as three high-risk cancer families. Compared to that in sporadic breast tumors (53.3%, 16/30) and healthy blood donors (6.7%, 2/30), we identified a total of 126 sequence alterations in 23/23 (100%) of familial breast cancer patients, including eight novel nucleotide variants. Among these changes, A to G at nt.263, T to C at nt.489, T to C at nt.310, TC insertion at nt.311, CA deletion at nt.522, and C to G at nt.527 were highly frequent ones. In addition, among three high-risk cancer families, we found that individuals affected with breast cancer harbored more mtDNA sequence variants in mtDNA D310 area than other affected family members. Together, our data indicate that sequence variants within the mtDNA D-Loop region are frequent events in Chinese familial breast cancer patients. Some of these nucleotide abnormalities, particularly those in D310 segment, might be involved in the breast carcinogenesis and could be included in a panel of molecular biomarkers for cancer susceptibility early-detection strategy.