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The high biodiversity of fish in the Neotropical region contrasts with scarce or biased studies on the mechanisms involved in the sex determination in members of this fauna. In this review, we attempted to compile the information available on determination, differentiation, and manipulation of sex for Neotropical species, with special focus on silversides and other two speciose groups, known as characins (Characiformes) and catfishes (Siluriformes). Currently, there is plenty of information available on chromosomal sex determination systems, which includes both male and female heterogamety with many variations, and sex chromosomes evolution at the macro chromosomal level. However, there is hitherto a blank in information at micro, gene/molecule levels and in research related to the effects of environmental cues on sex determination; most of reported studies are limited to silversides and guppies. In view of such a high diversity, it is critically necessary to establish key model species for relevant Neotropical fish taxa and also multi-disciplinary research groups in order to uncover the main patterns and trends that dictate the mechanisms of sex determination and gonadal differentiation in this icthyofauna. By increasing our knowledge on sex determination/differentiation with the identification of sex chromosome-linked markers or sex-determining genes, characterization of the onset timing of morphological gonadal differentiation, and determination of the environmental-hormonal labile period of gonadal sex determination in reference species, it will be possible to use those information as guidelines for application in other related groups. Overall, the strategic advance in this research field will be crucial for the development of biotechnological tools for aquaculture industry and for conservation of fish fauna from the Neotropical Region., (Copyright © 2018 Elsevier Inc. All rights reserved.)