The accumulation of single nucleotide polymorphisms (SNPs) in the displacement loop (D-loop) of mitochondrial DNA (mtDNA) has been described for various types of cancer, and their association with cancer risk and disease outcome has been extensively identified. In the present study, we investigated the association between age-at-onset and SNPs in the mitochondrial D-loop using a population-based series of esophageal squamous cell carcinoma (ESCC) patients. The SNP sites of nucleotides 16266 C/T were identified for their association with age-at-onset using the log-rank test. The age-at-onset of patients with the minor allele T genotype was significantly lower than that of patients with the C genotype at the 16266 site (p=0.036). Genetic polymorphisms in the D-loop are predictive markers for age-at-onset in ESCC patients. Accordingly, the analysis of genetic polymorphisms in the mitochondrial D-loop may help identify ESCC patient subgroups at high risk of early onset.