The aim of this study was to investigate mitochondrial 16S rRNA sequence variation to assess genetic structure and polymorphism of in four stocks of Marsupenaeus japonicus. The samples of M. japonicus were obtained from the East Sea and South Sea of Guangdong ( GD),Taiwan ( TW),Fujian ( FJ) and Zhejiang (ZJ) Province in China. The PCR technique was used to amplify the mtDNA 16S rRNA gene fragment. Four hundred and seventy bp nucleotide sequence of partial 16S rRNA was obtained,and 16 variable sites and 10 haplotypes were detected among all sequences. Nucleotide diversities in GD stock,TW stock,FJ stock and ZJ stock were 0. 000 8,0. 001 0,0. 005 1 and 0. 001 5 respectively. Each stock had shared haplotype and unique haplotype. F_st analysis showed that genetic difference existed among four stocks,and significant genetic differentiations were observed between FJ stock and other three stocks. The Kimura-2-paramter genetic distance calculated by the MEGA software between the stock of FJ and TW was the highest,up to 0. 003 2,while that between the stock of ZJ and GD was the lowest to 0. 000 9. NJ phylogenetic tree was constructed using 12 Penaeidae shrimp. These results will be important and useful for making scientific strategy for the family selective breeding and molecular marker assisted selective breeding.