Relationships among the species of the annual fish Cynolebias are unclear. An analysis of the variation and utility of osteological characters for phylogenetic analysis was done using cleared and double-stained specimens representing 21 species of Cynolebias. This analysis showed that some of the characters previously used to diagnose this genus and some of the species are polymorphic. Osteologically, Cynolebias can be diagnosed by the following synapomorphies: (1) triangular-shaped parietal, (2) vomer positioned ventral to the parasphenoid, (3) long ventral process of the dentary, (4) teeth on fourth ceratobranchial, and (5) teeth on first epibranchial. In addition, characters that help define some of the currently recognized species complexes were identified. Species in the ''antenori complex'' share at least five synapomorphies, such as ossified medial radials of dorsal and anal fins, four pectoral radials, ventral process of the maxillae enlarged, mesopterygoid long relative to the autopalatine, and proportion of cartilage in the basihyal. The ''bellottii complex'' is characterized by having a reduce basihyal and a deep urohyal, whereas species in the ''elongatus complex'' possess a caudal fin supported by four vertebrae and have unique modifications of jaw bones. The following osteological features are useful as diagnostic characters at the specific level: ( ) Two vertebrae supporting the caudal fin (C. nigripinnis); (2) cartilaginous pelvic bones (C. notatus); (3) short third postcleithrum and broad lateral process of the sphenotic (C. wolterstorffi); (4) thick third postcleithrum (C. gymnoventris); (5) crest on the parietal and reduced in the upper portion of the lacrimal (C. whitei); (6) anteriorly curved lacrimal (C. cheradophilus); (7) second dermosphenotic (C. bellottii); and (8) expansion of the ventral tip of the maxillae and long basyhial (C. constanciae).