The present study aimed to investigate gene mutations in the displacement‑loop (D‑loop) region of mitochondrial DNA (mtDNA) in patients with oral squamous cell carcinoma (OSCC) in order to examine the role of gene mutation in mtDNA in OSCC tumorigenesis. mtDNA was obtained from cancer tissues, paracancerous tissues and normal mucosal tissues of thirty patients with OSCC. The D‑loop region of the mtDNA was amplified using polymerase chain reaction, sequenced and then analyzed by Chromas software and BLAST to identify the mutation sites. Mutations in the D‑loop region were observed in the cancer tissue samples of eight out of thirty cases with OSCC, with a mutation rate of 27%. There were nine mutations in total, including one point mutation, two base deletions, three insertion mutations and three heterozygous mutations. In these mutations, base deletions were different from each other and heterozygous mutations did not have the same mutation form; however, the three insertion mutations were the same, consisting of an insertion of a C base. One case contained a T/A heterozygous mutation as well as base insertion of C. The eight cases with mutations in the D‑loop region consisted of three cases of tongue cancer, two cases of soft palate cancer, one case of floor of the mouth cancer, one case of oropharyngeal cancer and one case of lip cancer. This study demonstrated mutations in the mtDNA D‑loop region in OSCC cells; however, the association between occurrences of OSCC and mtDNA mutations requires further investigation.