The aim of the present study was to detect mutations in the coding genes of mitochondrial DNA (mtDNA) in three esophageal cancer cell lines and in tumor tissues obtained from 30 patients with esophageal cancer, to investigate the relationship between protein‑ and RNA‑coding gene mutations and esophageal cancer. mtDNA was extracted and the coding genes were sequenced and analyzed by comparing the sequencing results with the complete mitochondrial genome of Homo sapiens. The results revealed 39 mutations in the three esophageal cancer cell lines; the genes with the highest mutation frequencies included mitochondrially encoded cytochrome B (MT‑CYTB), NADH dehydrogenase 5 (MT‑ND5) and MT‑ND4 gene. A total of 216 mutations were identified in the 30 esophageal cancer tissues, including 182 protein‑coding mutations, of which MT‑CYTB and MT‑ND5 genes exhibited higher mutation frequencies. The results of the present study indicated that mutations in the coding genes of mtDNA in esophageal cancer cells may be related to the occurrence of esophageal cancer.