Anyphaenidae are diagnosed by the pattern of tracheal system, the forwardly advanced tracheal spiracle, and the lamelliform claw tuft setae. Cladistic analyses of the relationships of the anyphaenid genera are made from a selection of 9 exemplar taxa scored for 20 characters. In result, three subfamilies are proposed. Malenellinae subfam. n. is erected for Malenella nana gen. & sp. n. from southern Chile. This subfamily is considered as the sister group of all other anyphaenids. Anyphaeninae and Amaurobioidinae form a monophyletic group united by the orientation of the claw tuft setae and the grooved cymbial tip. Anyphaeninae are diagnosed by three characters associated with the advancement of the spiracle toward the epigastric furrow. Amaurobioidinae are diagnosed by the ingression of the median hematodocha in the male tegulum, and a characteristic secondary conductor. The relationships among the genera of Amaurobioidinae are discussed. The complex tracheal system of anyphaenids develops, during ontogeny, from a simple four-branched system. The genera Aysenia (from Clubionidae), Philisca (from Miturgidae) and Sanogasta (from Corinnidae) are transferred to Anyphaenidae: Amaurobioidinae.