Earlier studies of mitochondrial mutations in melanoma have focused on analysis of selected mitochondrial genes and the displacement loop (D-loop) region using conventional sequencing. In this study we use data from a whole mitochondria-sequencing array, the MitoChip v2.0, to characterize the mutations that are present throughout the mitochondrial genome. The mitochondrial genome of DNA derived from 14 fresh melanoma specimens and two melanoma cell lines, and autologous lymphocytes or immortalized B cells, respectively, were sequenced using the MitoChip v2.0. Paired comparative sequence analysis was carried out to define somatic mutations. Somatic mitochondrial DNA mutations were identified in 12/16 (75%) melanomas, compared with germline lymphocyte DNA. One hundred mutations were present among these 12 melanomas. A disproportionate number of mutations occurred in the D-loop. Furthermore, 9/16 (56.3%) melanomas carried mutations, which resulted in amino acid substitutions in functional genes. In the 10 samples carrying nicotinamide adenine dinucleotide dehydrogenase (ND) complex mutations, multiple mutations were present at a rate significantly greater than the expected frequency based on the size of ND complex genes (P=0.028, Fisher's exact test). Mitochondrial mutation is a frequent occurrence in melanoma. The high rate of missense mutations and the propensity for the ND complex implicate a role for alterations in mitochondrial respiratory function in melanoma carcinogenesis. Mutations of the noncoding D-loop are of unclear significance, but may be associated with alterations in transcription or replication. Further studies are needed to delineate the timing and functional significance of these mutations, and their role in the pathogenesis of this disease.