Genetic changes in mitochondrial DNA (mtDNA) have been detected in a variety of pathologic conditions including cancer. We hypothesized that malignant melanoma has genetic alterations in the displacement loop (D-loop) region and that these mtDNA alterations can be detected in blood as a circulating DNA melanoma marker. D-loop region from 20 melanoma cell lines, 12 metastatic melanoma specimens, and corresponding lymphocytes and plasma samples were sequenced using the CEQ 8000 XL Genetic Analysis System (Beckman Coulter). Nine of 20 (45%) melanoma cell lines and 5 of 12 (42%) melanoma specimens contained somatic mutations in the D-loop region of mtDNA. DNA alterations in the polycytosine tract (C-tract) of D-loop were detected in 6 of 20 (30%) cell lines and 2 of 12 (17%) specimens. Two of five paired plasma samples (40%) contained the same mutations as did melanoma specimens. In a comparison of lymphocytes and plasma of melanoma patients, 9 of 44 paired plasma samples (20%) contained at least one mutation compared to corresponding lymphocytes. Somatic mutations in the D-loop region of tumor and paired plasma did not correlate with the clinicopathological characteristics. However, circulating mtDNA alterations were more frequent in advanced disease. Studies indicate that circulating mtDNA mutations in the plasma of melanoma patients can be detected.