Gasterosteiformes including sticklebacks are extensively used for behavioral, physiological, ecological, and other comparative studies. However, the systematic position and phylogenetic relationships of the group are subject to much disagreement. The main objective of this study was to test recent hypotheses of relationships of Gasterosteiformes. The phylogenetic relationships of Gasterosteiformes was studied in an osteological examination of representatives of 48 genera in 41 families of eurypterygian fishes to hypothesize their systematic relationships. The outgroup comparison method was used for polarizing characters and cladistic methods for reconstructing systematic relationships of Gasterosteiformes. Monophyly of Acanthomorpha, Acanthopterygii, Percomorpha, and Smegmamorpha is supported. Mugiliformes (mullets) and Atherinomorpha (silversides, needlefishes, and pupfishes) are shown to be each other closest relatives. This study shows that Synbranchiformes (swamp eels and spiny eels) is the sister group of Gasterosteiformes and that Elassomatiformes (Elassomatidae, pygmy sunfishes) is the sister group of Synbranchiformes and Gasterosteiformes. To reflect the new phylogeny in a classification of Percomorpha, it is suggested that the series Percomorpha be divided into two subseries: Smegmamorphei and Percomorphei. Although no unique synapomorphy was found to unit all the Gasterosteiformes, support was provided that Gasterosteiformes (including Hypoptychidae and Indostomidae) is a monophyletic group. Based on the phylogenetic analysis and synapomorphies provided for the subgroups, three suborders in Gasterosteiformes are recognized: Hypoptychoidei, Gasterosteoidei, and Syngnathoidei.