The order Clupeiformesis in the Class Osteichthyes, and many of these species are of significant economicvalue. The mitochondrial cytochrome c oxidase subunit 1 (CO I) gene sequences of seven species pertainingto six genera in Clupeiformes collected from Chinese coastal waters were amplified using a polymerase chain reactiontechnique. The homologous sequences of other species of Clupeiformes from GenBank were also includedin this study. A total of 273 single sequences from 90 species pertaining to 41 genera in Clupeiformes were barcodedusing the CO I gene and then sequenced and compared to verify the feasibility of using DNA barcoding toidentify species. Clustal X and MEGA6.0 were used to calculate base composition, sequence substitutions, andgenetic distance. The results indicated that mean T, C, A, and G contents were 28.3%, 28.3%, 24.2%, and 19.2%,respectively, indicating that base composition was biased.The nucleotide sequences revealed base substitutions,including 71 conversion sites and 46 transversion sites. Most appeared on the third codon, including 62 conversionsites and 45 transversion sites. The second codon did not have conversions or transversions. The conversion andtransversion ratio reflects evolutionary rate; if the probability of transversion is greater, the conversion and transversionratio is smaller, and the evolutionary rate is faster. The evolutionary rate of the third codon was the fastest,followed by the first codon, and the second codon was the most stable. DNA barcoding can be used to identifyspecies if the distance between congeners is much larger than that between conspecifics and different individualsof the same species are clustered in the same phylogenetic tree branch. The congeneric and conspecificgeneticdistances were 0.131 and 0.003, respectively, and the congeneric distance was 41 times that of the conspecificdistance. However, some congeneric distances were too small or the conspecific distances were too large, leadingto different names for the same species and intraspecific differentiation. All conspecifics clustered together in thephylogenetic tree, except Dorosoma cepedianum and Dorosoma anale, suggesting that these may be the same species.The phylogenetic analysis showed that 97.8% of the fish were monophyletic. In summary, the DNA barcodesof clupeiform fish complied with the requirements for species identification, and the neighbor-joining tree basedon the CO I gene identified species more accurately. The phylogenetic results show that the CO I gene was suitablefor clarifying primary taxa and provided some reference value for the phylogenetic relationships of senior taxa.