Genetic variation and differentiation of four Marsupenaeus japonicus stocks collected from the coastal waters of southeast China were investigated based on 467 bp nucleotide sequences of mitochondrial 16S rRNA. Among 467 bp sequences, 45 variable sites (9.64%)were detected.. Comparisons of these 25 sequences revealed 10 haplotypes. The haplotype diversity (h) of Sanya stock was the highest(0.900±0.161). Also the nucleotide diversity (π) of SY stock was far higher than that of other three stocks and was ranged from 0.0007 (BH) to 0.0082 (SY). The Fst values calculated by the analysis of molecular variance (AMOVA ) ranged from 0.00 to 0945. Combined with other reported 16S rRNA gene sequences of M.japonicus, the neighbor-joining (NJ) tree of haplotypes suggested that there are three existed clades. One clade included ZJ, BH and HK haplotypes, all haplotypes from Hongkong to Taiwan coastal waters formed another clade, these two clades clustered first and belong to the reported varieties Ⅰand varietiesⅡ, respectively. Then these two clades clustered with the third clade (included four SY haplotypes). The nucleotides variation between Sanya clade and other two clades was very high (7.95%-8.35%). One new species (or varieties) may exist in Sanya waters. Further study should be conducted to get more evidences to verify this result. Results can offer genetic basis for resource management and further breeding program.