Acanthochiton rubrolineatus (Cryptoplacidae, Neoloricata, Polyplacophora) has a narrow distribution range along the seacoasts of China, the Korean Peninsula and Japan. We collected 238 samples from eight localities along the Chinese coast, and analyzed the genetic diversity and population structure with COI, 16S-rRNA and 28S-rRNA gene sequences. All analyses based on combined sequences of COI and 16S-rRNA suggested that there was evident genetic differentiation between the northern populations (YT, WH, DL, QD, LYG) and southern populations (ZS, YH, XM) of A. rubrolineatus. The haplotype distribution pattern and genetic diversity based on 28S-rRNA sequences also supported the genetic divergence between the two groups. Both groups had experienced population expansion after the ice age of Pleistocene, and an additional population bottleneck had happened in the southern group in recent history, which led to low genetic diversity of mitochondrial DNA and abnormally high diversity of nuclear DNA in this group. Our results suggested that the protection on A. rubrolineatus is necessary, and the northern and southern group should be protected separately.