Objective: To ascertain the variations of mitochondrion DNA (mtDNA) in mouse tumors and to inquire into the relationship between mutations of mtDNA and carcinogenesis. Methods: The variations of D-loop, ND3 and tRNAMet+Glu+Ile gene fragments of mtDNA from six tumor cell lines of mice were analyzed by PCR technology with restriction fragment length polymorphism analysis (polymerase chain reaction-restriction fragment length polymorphism, PCR-RFLP) and single strand conformation polymorphism analysis (SSCP-PCR) method. Results: ND3 and tRNAMet+Glu+Ile gene fragments of mtDNA from the tumors showed no variation in 27 endonuclease sites; D-loop of mtDNA from Hca-F had an additional endonuclease sites of Hinf Ⅰ in contrast to that of the inbred mouse. Deeply analyzed by PCR-SSCP, the D-loop of mtDNA was found to possess mutations in 4 of 6 tumors. Conclusion: D-loop is the hot spot of tumor mtDNA mutation which can act as contributors to the carcinogenic process.