Kryptolebias marmoratus is an important experimental fish, and is considered to represent the only vertebrate species comprising self-fertilizing hermaphroditic individuals. Subsequent to the discovery of this unusual mode of reproduction, approximately 50 years ago, K. marmoratus has been the focus of a series of studies. However, little is known about the evolution of this rare reproduction mode, and data on the biology of closely related species are still unavailable. The present study aimed to histologically analyse the gonads of three K. marmoratus congeners (i.e. Kryptolebias ocellatus, Kryptolebias caudomarginatus, and Kryptolebias brasiliensis) to check the distribution of features related to hermaphroditism that are useful for forming hypotheses about the origin and evolution of the self-fertilization mode of reproduction through the available phylogenies. The data obtained demonstrate that populations of K. caudomarginatus consist of males and hermaphrodites, which supports the hypothesis that hermaphroditism arose at the base of the clade containing K. caudomarginatus, K. marmoratus, and K. ocellatus as a first step towards a more advanced condition, uniquely shared by both K. marmoratus and K. ocellatus, in which males are rare or absent in natural populations, with the subsequent occurrence of self-fertilization. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 99, 344–349.