The accumulation of single nucleotide polymorphisms (SNPs) in the displacement loop (D-loop) of mitochondrial DNA (mtDNA) has been described for different types of cancers, and the association of these SNPs with cancer risk and disease outcome has been exhaustively studied. We sequenced a region of approximately 1 kb flanking the majority of the D-Loop in the DNA from the blood of breast cancer patients and the controls to identify cancer risk-associated D-loop SNPs. The D-loop region of mtDNA was sequenced from 92 sporadic breast cancer patients, 60 familial breast cancer patients and 41 relatives, and 93 healthy controls. Paired and unpaired Student's t tests were used as appropriate to determine the differences in SNP distribution within the D-loop region and in the number of SNPs per patient among the groups. The chi (2) test was used to analyze dichotomous values, such as the presence or absence of an individual SNP among each group, and the clinical characteristics between every two groups. The distribution frequencies of 315C/Cinsert, 524C/del, 16247A/del, 16248C/del, 16249T/C, 16257C/A, 16258A/del, 16259C/del, 16262C/del, 16268C/del, 16279C/del, 16280A/del, 16297T/C, and 16300A/del were significantly different between sporadic breast cancer patients and the normal controls. The SNP sites at nucleotides 310, 315, and 16362 were identified as cancer risk-associated SNPs specific for familial breast cancer. The N haplogroup, defined as 489T, was identified as a specific risk-associated SNP for families of breast cancer patients by comparing familial breast cancer patients with their relatives. The analysis of genetic polymorphisms in the D-loop may help to predict cancer risk for familial breast cancer and thereby help to detect and refine therapeutic decisions earlier.