Objective Spirometra erinaceieuropaei sparganum larvae were isolated from different species of snake and identified by morphology and molecular biology. The polymorphism of partial mitochondrial NADH dehydrogenase subunits 1 gene(pnad1) amplified from these isolated spargana was analyzed for its potential as a species marker. Methods Eleven sparganum larvae of S. erinaceieuropaei were isolated from different species of wild snake [Elaphe carinata (3), Zaocys dhumnade (2), Naja (1), Ptyas korros (2), Ptyas mucosus (2) and Bungarus multicinctus(1)] in Yongzhou, Chenzhou, Yueyang of Hunan Province and identified by morphology and DNA sequence. The pnad1 gene was amplified from these spargana isolated from different snakes and sequenced. The obtained sequences were aligned to determine their nucleotide variations and compared with their homologues from other tapeworms, and then a phylogenetic tree was constructed accordingly using MEGA7.0. Results The 11 larvae were identified as Spirometra erinaceieuropaei spargana by their morphology under microscopy and DNA sequence. The pnad1 gene was amplified from these collected spargana with size of 648 bp. The sequence results showed that the amplified pnad1 sequences were almost identical with 0-1.4% variation, but with 97.8%-99.3% sequence identity with different isolations of Spirometra erinaceieuropaei deposited in GenBank, and with less than 74.3% identity with pnad1 sequence of other tapeworms. Constracted phylogenetic tree based on the pnad1 gene sequences showed that the Hunan isolates were clustered in the same clade of Spirometra erinaceieuropaei, genetically closer to Dibothriocephalus latus and Hymenolepis nana, but further to other tapeworms. Conclusion The sparganum larvae isolated from different species of snakes in Hunan in the present study were identified as Spirometra erinaceieuropaei, and the mitochondrial pnad1 gene can be used as a genetic marker to identify tapeworm species.