Abstract: DNA Barcoding is becoming more and more attractive to biologists as a new method for species identification.The core idea of DNA barcoding is based on the fact that species can be differentiated by short pieces of standard gene sequences.It is very important to choose appropriate genetic marker for species identification.MitochondrialCO1 gene sequence was a popular marker of DNA barcoding for species identification in the kingdom of animals.In this paper,we discussed the validation ofCO1 gene sequence to efficiently identify the species of genus Culter.Original nucleotide sequence data of partial mitochondrialCO1 gene,about 816 base pairs,were collected from 4 species of Culter and totally 32 specimens.Sequences analysis was conducted by using MEGA3.1.Genetic variation among species and variation within species were calculated,and it was found that pairwise genetic distance among 4 species was more than 2%,and obviously higher than the distance within a species.Phylogenetic analysis was carried out with different methods,including neighbor-joining,maximum parsimony,maximum likelihood and bayesian analysis.The topology of each final trees revealed that all individuals of each species formed a strong monophyletic group.Specie identification withCO1 sequences in Culter supported the taxonomy based on morphological characters,and it was proposed thatCO1 sequences could be a probable marker to resolve the phylogenetic relationship of the genus.The conclusion of our research suggested that mitochondrialCO1 gene was a valid DNA barcoding gene for specie identification in genus Culter.