A phylogenetic analysis based on morphological characters indicates that Leptolebias Myers, 1952, a genus of small killifishes highly threatened with extinction, from Brazil, is paraphyletic. As a consequence, Leptolebias is restricted in this study to a well-supported clade that includes Leptolebias marmoratus (Ladiges, 1934), Leptolebias splendens (Myers, 1942), Leptolebias opalescens (Myers, 1942), and Leptolebias citrinipinnis (Costa, Lacerda & Tanizaki, 1988), from the coastal plains of Rio de Janeiro, and Leptolebias aureoguttatus (Cruz, 1974) (herein redescribed, and for which a lectotype is designated) and Leptolebias itanhaensis sp. nov., from the coastal plains of Sao Paulo and Parana, in southern Brazil.Leptolebias is diagnosed by three synapomorphies: a caudal fin that is longer than deep, a single anterior supraorbital neuromast, and dark pigmentation that does not extend to the distal portion of the dorsal fin in males. A key is provided for the identification of species of Leptolebias. Three species formerly placed in Leptolebias, Leptolebias minimus (Myers, 1942), Leptolebias fractifasciatus (Costa, 1988), and Leptolebias cruzi (Costa, 1988), are transferred to Notholebias gen. nov., which is hypothesized to be the sister group to the clade comprising Leptolebias, Campellolebias Vaz-Ferreira & Sierra, 1974, and Cynopoecilus Regan, 1912. Notholebias gen nov. is diagnosed by two synapomorphies: a narrow basihyal and the presence of iridescent bars on the caudal fin in males; and three features interpreted as plesiomorphic, but not occurring in Leptolebias, Campellolebias, or Cynopoecilus, the presence of dermosphenotic, well-developed contact organs on the pectoral fin in males, and an opercular region with red bars in males. ‘Leptolebias’leitaoi, a species from Bahia, in north-eastern Brazil, is considered as having an uncertain phylogenetic position, as all known preserved material is presently lost, and the species may be extinct. © 2008 The Linnean Society of London, Zoological Journal of the Linnean Society, 2008, 153, 147–160.