Population genetic structure and historical demography of Chinese horseshoe crab (T. tridentatus) along southeast coast of China were inferred from the sequence data of mitochondrial cytochrome c oxidase subunit I (COI) fragment. The sequence analysis for 964 bp COI fragment was conducted in 28 individuals collected from five localities: Ninghai in Zhejiang Province, Meizhou and Zhangpu in Fujian Province, Beihai of Guangxi Zhuang Autonomous Region and Danzhou of Hainan Province. Sequence variation was relatively low with a total of seven transitions observed. In all localities, :Haplotype H3 was the dominant type observed among eight haplotypes defined previously, and was at the center of radiation in Median-Joining network. The prolonged star-like network suggests a signature of population expansions. The level of diversity was low in total, with haplotype diversity (H-d) being equal to 0.765 and nucleotide diversity (pi) being equal to 0.001 18, respectively. The genetic structure analysis revealed the significant genetic difference between Ninghai and Danzhou populations. Both mismatch distribution analysis and Fu's Fs test provided consistent inference of historic population expansion. The low genetic diversity of horseshoe crab observed along China coast indicated that urgent measures should be taken to protect this rare marine animal.