Phylogenetic relationships of Gasterosteiformes were studied using osteological examination of representatives of 11 families of gasterosteiform fishes, as ingroups, and 5 families of other smegmamorph fishes (Atheriniformes, Elassomatiformes, and Synbranchiformes), as outgroups. Based on phylogenetic analysis of 110 informative osteological characters, nine synapomorphies were found to unite all Gasterosteiformes and support was provided to the hypothesis that the order Gasterosteiformes (including Hypoptychidae and Indostomidae) is a monophyletic group. Furthermore, based on the synapomorphies provided for the subgroups, three suborders in Gasterosteiformes are recognized: Hypoptychoidei, Gasterosteoidei, and Syngnathoidei.