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Spermatogonial stem cells (SSCs) also known as germ stem cells (GSCs) are the basis of spermatogenesis process in the testis. Furthermore, they are also valuable cells with different applications in developmental biology, transgenesis technology, and clinic. Understanding the new findings related to the cell and molecular biology of SSCs and the methods for isolation and maintenance of these cells are important and essential for their applications in medicine to treat some infertility problems and also in biotechnology to produce transgenic animals. The present review was conducted to describe the cell and molecular basis of development, self-renewal, and differentiation of mammalian and poultry SSCs in vivo (natural niche) and in vitro. Moreover, this study represents specific molecular markers to characterize SSCs. We also introduce methods to isolate, cultivate and enrich these cells, which are important for their applications. Finally, the significance of SSCs in different fields and their practical perspectives, and also the differentiation potential of other stem cells into spermatogonial- and spermatic-like cells are discussed.