With the rapid development of modern molecular biology tools, especially DNA barcoding technology, a rapid, accurate and efficient identification method for species of zooplankton resting eggs is possible. In this study, zooplankton resting eggs, including copepods,cladocerans and rotifers in the surface sediments of Lake Liuye (Changde), a typical urban lake in Lake Dongting Basin, were studied. Using DNA barcode technology for species identification, 101 valid DNA sequences were successfully obtained from 191 resting eggs, and the identification success rate was 53%. According to the NCBI database comparison, 9 families, 12 genera and 11 species of resting eggs were successfully identified, and 6 samples were only identified to the genus or family level. The inter-species genetic distance of the three taxa was 68 times that of the intra-species genetic distance, indicating that the cytochrome oxidase Ⅰ (COⅠ) gene could be used for effective species identification of zooplankton resting eggs. Through the phylogenetic analysis by Neighbour-Joining tree, it was found that the identified species and their reference sequences were clustered into one branch, and all different species were clustered into independent branches respective. Therefore, different species could be effectively distinguished. The above results all indicate that the mitochondrial COⅠ gene, as a DNA barcode, can be used to identify the species of zooplankton resting eggs in sediment of lakes.