The present study aimed to detect the mutation characteristics of mitochondrial DNA (mtDNA) in Eca109 of Ec9706 cells, and to investigate their association with the nuclear genome (nDNA), thus providing a basis for gene targeting therapies for esophageal squamous cell carcinoma (ESCC). In vitro‑cultured Ec9706 and Eca109 cells were analyzed the changes of single‑nucleotide polymorphisms (SNPs), insertions/deletions (INDELs), copy number varia-tion, and structure variation (SV) of their genome by high‑throughput sequencing. The loci with SV on chromosome 1‑12 of the two ESCC cell lines were ≥5% of the mtDNA, but SV on chromosome 13‑22, X and Y was ≤3%; >40% of loci exhibited gain or loss; intergenic loci with INDEL changes and SNP features accounted for the majority of mutations. The affected genes encoded proteins including nDNA‑encoding intra‑mitochondrial‑transporting proteins, ATP energy generation‑associated proteins and mitochondrial electron respiratory chain proteins, and these proteins were all nucleus‑encoded mitochondrial proteins. The transcription, duplication, and translation of the abnormally expressed mtDNA in Ec9706 and Eca109 cells were closely associated with disorders of nuclear DNA products.