The identification and isolation of spermatogonial stem cells in Nile tilapia is crucial to the development of high-quality transplantation techniques as well as to understand the regulation of spermatogonia in vivo. However, specific molecular markers for spermatogonial stem cells have not yet been studied in detail. Taking advantage of this species as a good experimental fish model, we investigated the isolation and culture of spermatogonial stem cells from tilapia testis. Also, the expression of Gfra1, Notch1, and CD44 as molecular markers were studied using real-time RT-PCR, immunohistochemistry, and flow cytometry analysis. The result showed that enzymatic digestion, percoll gradient, and differential adhesion are suitable for the isolation of an enriched population of spermatogonial stem cells from tilapia´s testis. Moreover, the isolated spermatogonia expressed Gfra1, Notch1, and CD44 in vivo and in vitro. In conclusion, Gfra1, Notch1, and CD44 can be considered as good markers for spermatogonial stem cells in fish which may be effective to characterize and isolate spermatogonial stem cells from the Nile tilapia.