DNA barcoding offers a rapid and accurate assessment of proper labeling for the identification of species,which depends on genetic diversity being markedly lower within than between species.Here,we investigated the performance of DNA barcodes in a sample comprising of 29 species of the family Syngnathoidei,an unique group of fish with male pregnancy.A total of 1,002 DNA barcodes using 649 base-pair fiagrnents of the cytochrome c oxidase subunit Ⅰ(COI)gene were generated.Most species were associated with a distinct DNA barcode,and seven of the COI barcode clusters represented the first species records submitted to the BOLD and GenBank databases.Phylogenetic relationship was apparent from the data during the species identification.In the Bayesian phylogenetic tree of COI sequences,two major clusters of Gastrophori and Urophori were displayed,which could also be classified in morphology.We found that DNA barcoding displayed the morphological barriers among some fish species in this group.The tail-brooding Hippichthys cyanospilus and trunk-brooding Syngnathoides biaculeatus were reversely clustered together with the Gastrophori and the Urophori,respectively.Both seahorse and pipefish sequences showed high frequency of nucleotide substitutions,and the probability in the sequences of all pipefish is higher than that of of all seahorses.However,we identified no signal of positive selection on the COI gene in all data set.Our results supported DNA barcoding as an efficient molecular tool for the study of relationships among,monitoring and conservation of syngnathid species.