The Characidae is the most diverse family of Neotropical fishes, currently encompassing more than one thousand valid species. Some subgroups within this family still lack phylogenetic definitions, being diagnosed on the basis of combination of characters, a common procedure in pre-cladistic studies. Agoniatinae, currently composed by two valid species, Agoniates anchovia and A. halecinus, is one of them. In the present study the Agoniatinae is redefined using a phylogenetically oriented comparative survey that included the two Agoniates species plus 114 species representing all the major clades of the Characidae and their closest relatives. Six derived morphological characters are identified as synapomorphies for the Agoniatinae: deep notch on the posterior region of the maxilla joining the ventral margin of the infraorbital 2; dentigerous portion of premaxilla shorter than the ascending process of this bone; dentary canine preceded by tricuspid teeth; ventral margin of the urohyal markedly convex; base of the uppermost ray of the ventral lobe of the caudal fin much expanded, being as deep as the distal margin of hypural 2; and levator arcus palatini muscle with a posterodorsal bundle of fibers attaching to the dorsal face of the sphenotic spine.