Somatic mutations have been identified in mitochondrial DNA (mtDNA) of various human primary cancers. However, their roles in the pathophysiology of cancers are still unclear. In our previous study, high frequency of somatic mutations was found in the D-loop region of mtDNA of hepatocellular carcinomas (HCCs). In the present study, we examined 44 HCCs and corresponding non-cancerous liver tissues, and identified 13 somatic mutations in the coding region of mtDNAs from 11 HCC samples (11/44, 25%). Among the 13 mtDNA mutations, six mutations (T6787C, G7976A, A9263G, G9267A, A9545G and A11708G) were homoplasmic while seven mutations (956delC, T1659C, G3842A, G5650A, 11032delA, 12418insA and a 66bp deletion) were heteroplasmic. Moreover, the G3842A transition created a premature stop codon and the 66bp deletion could omit 22 amino acid residues in the NADH dehydrogenase (ND) subunit 1 (ND1) gene. The 11032delA and 12418insA could result in frame-shift mutation in the ND4 and ND5 genes, respectively. The T1659C transition in tRNA(Val) gene and G5650A in tRNA(Ala) gene were reported to be clinically associated with some mitochondrial disorders. In addition, the T6787C (cytochrome c oxidase subunit I, COI), G7976A (COII), G9267A (COIII) and A11708G (ND4) mutations could result in amino acid substitutions in the highly conserved regions of the affected mitochondrial genes. These mtDNA mutations (10/13, 76.9%) have the potential to cause mitochondrial dysfunction in HCCs. Taken these results together, we suggest that there may be a higher frequency of mtDNA mutations in HCC than in normal liver tissues from the same individuals.