Similar to mammals, spermatogenesis in fish is initiated by spermatogonial stem cells (SSCs) which either self-renew or gradually differentiate to produce mature sperm. SSCs are located in a particular testis microenvironment called SSC niche, formed by Sertoli and peritubular myoid cells, the basement membrane and other cellular components/factors from the intertubular compartment that regulate SSCs maintenance and fate. Considering the great variation in testis structure/arrangement across fish species, the study of the niche components is crucial to understand SSCs physiology. Additionally, the germ cell transplantation technique, which has been applied to fish in the last decade, is a unique approach to elucidating important functional aspects of SSCs biology such as: (i) the capacity of SSCs to colonize the testis of recipient species (syngeneic and xenogeneic transplantation) giving rise to donor sperm; (ii) the plasticity of these cells, considering that spermatogonia and oogonia can be derived from SSCs collected from the opposite sex; and (iii) the possibility of genetically manipulating SSCs before transplantation to produce transgenic fish. However, fish SSC isolation and characterization has been limited so far by the lack of specific molecular markers for these cells. Therefore, various research groups are currently investigating specific SSCs markers and, up to date, few proteins have been identified in different spermatogonial populations from distinct fish species (e.g. Notch1, Ly75, Plzf, Oct-4, SGSA-1). Furthermore, the development of a fish SSC culture system would allow the investigation of important regulatory aspects of the SSC physiology in well-defined conditions as well as to in vitro amplify these rare cells. Overall, the study of SSC physiology, niche and transplantation in fish has opened up new scenarios for the development of aquaculture and reproductive biotechnologies such as germplasm conservation of endangered or commercially important species and the possibility of generating transgenic fish