Somatic mutations of mitochondrial DNA (mtDNA) have been reported in different types of cancers and are suggested to play roles in metastasis, cancer development and response to anticancer agents. To predict potential roles of mtDNA alterations in colorectal cancer, we determined the entire mtDNA sequence of eleven human-derived colorectal cancer cell lines and compared with the revised Cambridge Reference Sequence to identify nucleotide alterations. Four homoplasmic and six heteroplasmic alterations were found to be novel. Among them, homoplasmic G6709A (MT-CO1) and G14804A (MT-CYB) alterations cause amino acid changes in the highly conserved residues. Heteroplasmic G1576A (MT-RNR1) and G2975A (MT-RNR2) alterations are expected to make the stem structure of mitochondrial ribosomal RNAs unstable. These nucleotide alterations are candidates that could play important roles in cancer.