The mitochondrial DNA (mtDNA) 4977-bp deletion (ΔmtDNA⁴⁹⁷⁷ mutation) is one of the most frequently observed mtDNA mutations in human tissues and may play a role in carcinogenesis. Only a few studies have evaluated ΔmtDNA⁴⁹⁷⁷ mutation in breast cancer tissue, and the findings have been inconsistent, which may be due to methodological differences. In this study, we developed a quantitative real-time PCR assay to assess the level of the ΔmtDNA⁴⁹⁷⁷ mutation in tumor tissue samples from 55 primary breast cancer patients and 21 patients with benign breast disease (BBD). The ΔmtDNA⁴⁹⁷⁷ mutation was detected in all of the samples with levels varying from 0.000149% to 7.0%. The ΔmtDNA⁴⁹⁷⁷ mutation levels were lower in tumor tissues than in adjacent normal tissues in both breast cancer and BBD subjects. The differences, however, were not statistically significant. No significant difference between breast cancer and BBD patients was found in the ΔmtDNA⁴⁹⁷⁷ mutation levels of tumor tissues and adjacent normal tissues. The ΔmtDNA⁴⁹⁷⁷ mutation levels were not significantly associated with clinicopathological characteristics (age, histology, tumor stage, and ER/PR status) in breast cancer or BBD patients. These results do not support the notion that the mitochondrial DNA 4977-bp deletion plays a major role in breast carcinogenesis.