BACKGROUND: Mitochondrial DNA (mtDNA) mutations occur in head and neck squamous cell carcinoma (HNSCC) and are most frequently detected in the displacement-loop (D-loop) region. The D-loop is considered to be important because it controls mitochondrial gene expression and mtDNA replication. There is currently no evidence that mtDNA mutations can be used as prognostic or predictive biomarkers in HNSCC. METHODS: We used denaturing high performance liquid chromatography to screen the entire mitochondrial genome of six oral squamous cell carcinoma-derived cell lines and then focused on detecting D-loop abnormalities in 34 HNSCC tissue samples. RESULTS: Mitochondrial DNA mutations are not ubiquitous in HNSCC because only half of the cell lines had detectable mtDNA abnormalities following screening of the entire mitochondrial genome and only 18% (6 of 34) of tissue samples had D-loop mutations. There was no correlation between D-loop mutations and determinates of clinical outcome; specifically, tumour stage and the expression of hypoxia-inducible genes included in a highly prognostic hypoxia metagene. CONCLUSIONS: Taken together, these data suggest that mtDNA D-loop mutations are stochastic events that may not significantly influence the biology of HNSCC and supports the hypothesis that mtDNA mutations in cancer represent bystander genotoxic damage as a consequence of tumour development and progression.