Objective To explore the feasibility of identifying clonal origin of hepatocellular carcinoma (HCC) by analyzing the mitochondrial DNA D-Loop region variations. Methods Forty-two patients with a total of 112 HCC nodules consequentially hospitalized for radical resection of HCC in the department of hepatobiliary surgery of the First Affiliated Hospital of Guangxi Medical University from April 2004 to August 2007 were included for study group (multinodular HCCs). Control group included 20 cases of HCC (40 samples) hospitalized in the same period that consisted of two sub-groups: control groupⅠconsisted of 16 cases of single nodular HCC that each had two pieces of inconsecutive tumor tissues and control groupⅡconsisted of 4 cases of HCC with portal vein tumor embolus whose tumor tissues and portal vein tumor embolus were collected simultaneously. Normal control included 5 patients who were donors for liver transplantation or underwent liver trauma without any liver disease. Polymerase chain reaction (PCR) and direct sequencing were applied to study the mtDNA D-Loop region. The sequences of multinodular lesions were compared among different groups. Results For all the 42 cases of the study group, basic group variations appeared in 131 sites (131/1 122, 11.7%, the number 1 122 was the length of mtDNA D-Loop) with point mutation in 15 sites, insert in 9 sites, and deletion in 16 sites. And of all the variations in the study group, 98 were polymorphism. In study group, 20 cases were categorized as multicentric occurrence (MO) based on their variant mtDNA D-Loop sequences in each nodule from the same patient. And 22 cases were characterized as intrahepatic metastasis (IM) based on the identical mtDNA D-Loop sequences found in each nodule from the same patient. In all 20 cases in the control group, the inconsecutive tumor tissues or the portal vein tumor embolus and original tumors shared identical mtDNA D-Loop sequences. For the normal control group, basic group variations appeared in 14 sites, and they were all polymorphism including a new polymorphism (NT 479 Agt;G). Conclusions There is a high rate of changes in mtDNA D-Loop region. And our study speculates a novel discrimination of MO and IM origins among multinodular HCCs using PCR and direct sequencing of the mtDNA D-Loop sequences.