We investigated the microsatellite alterations in mitochondrial DNA (mtDNA) and in TP53 in thoracic esophageal squamous cell carcinomas (TESCC). Using laser microdissection, 66 paired non-cancerous esophageal muscles, non-cancerous esophageal mucosa, cancerous TESCC nests plus 35 metastatic lymph nodes harvested from 66 resected esophagi of TESCC patients were subjected to DNA extraction. D310 and D17S960 were chosen as markers to address microsatellite alterations in mtDNA, including changes in copy number and homoplasmic/ heteroplasmic mutations of mtDNA, and in TP53, including loss of heterozygosity (LOH) and microsatellite instability (MI). From non-cancerous esophageal mucosa to cancerous TESCC nests and then metastatic lymph nodes, a trend of homoplasmic D310 mutation (10.6, 25.8, 31.4%; P=0.023), an ever increase of mtDNA copy ratios (0.892, 1.128, 1.183; P=0.018) and an elevated incidence of TP53 LOH (19.7, 34.8, 37.1%; P=0.010) were observed. From T1, T2, T3 to T4 TESCC, the incidence of TP53 LOH (12.5, 16.7, 34.8, 52.2%; P=0.011) was increased, in a stepwise fashion. Furthermore, we observed an association of TP53 LOH with an increased mtDNA copy ratio (P=0.022) and TP53 MI with heteroplasmic D310 mutation (P=0.069) in TESCC. Concurrent and associated microsatellite alterations in mtDNA and in TP53 in TESCC support the cancer clonal expansion theory and imply a possible relationship between the mitochondria and p53 in TESCC.