Sequence variability in three mitochondrial DNA (mtDNA) regions, namely cytochrome c oxidase subunit 3 (cox3), NADH dehydrogenase subunits 1 and 4 (nad1 and nad4) in Spirometra erinaceieuropaei spargana from different geographical regions in China was examined. A portion of each of the cox3 (pcox3), nad1 (pnad1) and nad4 genes (pnad4) were amplified separately from individual S. erinaceieuropaei spargana by polymerase chain reaction (PCR). Representative amplicons were subjected to sequencing in order to estimate sequence variability. The sequences of pcox3, pnad1 and pnad4 were 541, 607 and 847 bp in length, respectively. The A+T contents of the sequences were 68.39–68.76% (pcox3), 63.76–64.91% (pnad1) and 67.18–67.77% (pnad4), respectively, while the intra-specific sequence variations within each of the S. erinaceieuropaei spargana were 0–1.5% for pcox3, 0–2.8% for pnad1 and 0–2.7% for pnad4. Phylogenetic analysis using neighbour joining (NJ), maximum likelihood (ML) and maximum parsimony (MP) methods, indicated that all the spargana isolates in Hunan Province represented S. erinaceieuropaei. These findings demonstrated clearly the usefulness of the three mtDNA sequences for population genetics studies of S. erinaceieuropaei spargana of human and animal health significance.