In order to study the population genetic structure and germplasm resources of the Sipunculus nudus. mtDNA control region sequences of S. nudus of six geographic populations along the coast of Beibu Gulf (Beihai, Zhanjiang, Qinzhou, Fangchenggang, Danzhou of China and Haiphong of Vietnam) were analyzed. Results showed that a total of 93 control region sequences with 107 variable sites and 85 haplotypes were obtained, and an evident AT-skew was found in mtDNA control region sequences of S. nudus; the haplotype diversity (H_d) and nucleotide diversity (P_i) were 0.998 and 0.018 89 respectively. The NJ (Neighbor Joining) tree base on haplotype showed that the six populations presented no obvious branches, and all haplotypes were randomly distributed in the haplotype network. The range of fixation index (F_st) of different populations was -0.018 13-0.028 05, and the genetic differentiation among populations was not obvious. The results of AMOVA revealed that the genetic variance mainly occurred within populations (99.74%). In terms of the negatively selective neutrality test and mismatch distribution of pairwise, we could suggest that a population expansion occurred in S. nudus, and the estimated time was about 1 710 000 years ago. The results indicated that there was high genetic diversity in S. nudus and existed gene exchange among populations, and the estimated population expansion could occur during the Early Pleistocene.