Sipunculus nudus resources have been seriously damaged in the recent years due to overfishing and habitat destruction. Protecting natural resources of S. nudus has become more and more urgent. In this paper, genetic structure and population differentiation of S. nudus from 6 localities along the coasts of China were analyzed based on sequences of mitochondrial cytochrome b gene. Complete mitochondrial cytb sequences from 203 individuals of S. nudus were analyzed, 299 polymorphic sites and 128 haplotypes were identified and the nucleotide diversity and haplotypes diversity were 0.0488 and 0.9773, respectively. The variation among populations (accounting for 88.4%) was the main source of variation. According to differentiation index (F_st) in populations of S. nudus, the 5 populations in the south of Yantai showed no significant differences or low differences, with F_st<0.15. There were high differences between Yantai and other 5 populations, with F_st>0.15. Fu’s Fs and Tajima’s D were negative. Figures of mismatch distribution were unimodal. Haplotype network was radial structure centering on main haplotype. NJ phylogenetic tree of S. nudus based on 203 sequences of cytb of mtDNA revealed that these 6 populations were divided into 2 clades, one clade was Yantai population and the other clade included the rest 5 populations in the south of Yantai. All these results revealed that S. nudus population had experienced a large-scale population expansion in history, and the relatively low genetic diversity of S. nudus now suggested that the ability of S. nudus to adapt to environmental changes was poor and some measures should be taken to protect S. nudus resources. Phylogenetic tree suggested Yantai population was distinct from the other 5 populations, but whether Yantai population has become a cryptic species still needs further research.