As a routine and important polymorphic marker, mitochondrial cytochrome oxidase subunit Ⅰ (COⅠ) gene was used to analyze the genetic diversity for two populations of Oratosquilla oratoria from Shenzhen and Shanwei coast of Guangdong Province. The result revealed that 21 of the 592 bp (3.55%) nucleotides were variable, among which 18 and 3 sites were identified as transition and transversion respectively. The substitution saturation analysis indicated that COⅠ gene was not satured in O.oratoria and could be used as an effective molecular tool for population genetic studies. Totally, 15 haplotypes were detected. The nucleotide diversity and haplotype diversity were 0.007 55 and 0.971 respectively. The fixation index (FST) was 0.036 between two populations, suggesting little differentiation between them. There was no geographic clustering of haplotypes in the UPGAM tree and the network. Genetic distances among haplotypes ranged from 0.047 6 to 0.428 6, with an average value of 0.214 1. In summary, a high level of genetic diversity and little genetic differentiation are observed in O.oratoria from the eastern coast of Guangdong province.