DNA barcodes can be used to identify existing biological groups and unknown biomaterials through shorter DNA sequences using polymerase chain reaction (PCR). The mitochondrial cytochrome coxidase I (COI) gene is one of the commonly used DNA barcoding genes. In the present study, to analyze the effectiveness of the COI gene for DNA barcoding in mollusk evolution, 353 sequences of 60 species belonging to 13 orders and 33 families were obtained by PCR amplification for clustering analysis. A neighbor-joining (NJ) phylogenetic tree was constructed by Mega, and the genetic evolution of seven species from different populations was also analyzed. The results showed that the universal primers of the COI gene were highly versatile for most shellfish. Although the amplification efficiency of primers in Pterioida was only 10%, it reached 82.7% for the individuals. Except for the evolutionary status of eight species, such as , the clustering relationships of other species were similar to results obtained using traditional methods. Based on the clustering analysis of 7 species from 26 geographical groups, it was found that the COI gene generally clustered the different geographical groups of the same species first, and some individuals in the population or group had clustering disorder. In conclusion, the COI gene could be applied to the identification and phylogenetic studies of shellfish species to a certain extent, which would enhance the role of the COI gene in practical applications of species identification, and provide reference data for the construction of shellfish DNA barcoding databases.