The previous morphology-based taxonomic frameworks within the family Syngnathidae had emphasized the significance of the male brood pouch and reproductive biology in defining the group. However, several different hypotheses had been proposed by different investigators. This study has been carried out to determine the phylogenetic relationships among 19 species belonging to the order Syngnathiformes with three Gasterosteiformes species as outgroup taxa by using the mitochondrial cytochrome b DNA sequences. Phylogenetic analyses based on neighbor-joining distance, maximum parsimony, minimum evolution and maximum likelihood method strongly supported that the family Syngnathidae, the suborder Syngnathoidei and the order Syngnathiformes were all monophyletic group. Although much of previous morphological analyses were supported by our molecular data, there were some significant discrepancies between molecular and morphological work. Such an interesting result was that the weedy seadragon (Phyllopteryx taeniolatus) strongly grouped together with the New Zealand pot-belly seahorse (Hippocampus abdominalis). Considering the markedly different brooding structure between them, this unexpected result might be explained whether by multiple independent origins of brooding structure or by hybridization between the female Hippocampus and other syngnathid species having individual membranous egg compartment. In addition, the suborder Aulostomoidei was paraphyletic group because the shrimpfish (Aeliscus strigatus), belonging to the family Centriscidae, always grouped together with the family Syngnathidae as a sister taxon.