Objective: To investigate the prognostic correlation between mitochondrial DNA (mtDNA) content and colorectal cancer. Methods: 117 patients with colorectal cancer were selected and clinicopathologic data were collected. RT-qPCR was used to determine the mtDNA content of cancer tissues and paracancerous tissues. The correlation between mtDNA content and various prognostic indicators was investigated. ROC (Receiver Operating Characteristic) curves were plotted, and DFS (Disease Free Survival) curves were plotted based on cut-off values. Risk factors associated with DFS were analyzed by univariate and multivariate Cox regression analyses. Results: There was no significant difference in mtDNA content in tumor tissues compared to paraneoplastic tissues(P=0.432). Low mtDNA content was associated with colon tumor, poor differentiation, advanced TNM stage, and lymph node metastasis(P<0.05). The ROC curve indicated that the mtDNA content of 500.699 could be used as a cutoff value. Univariate and multivariate Cox analyses showed that mtDNA content below 500.699 (HR=4.285, 95%CI: 1.938-9.475) and poorly differentiated tumors (HR=2.886, 95%CI:1.428-5.835) were independent risk factors associated with DFS. Conclusion: Tissue mtDNA content is associated with clinicopathologic features in patients with colorectal cancer, and low mtDNA content is an independent risk factor associated with prognosis.