Spermatogonial stem cells (SSCs) are at the foundation of the highly productive spermatogenic process that continuously produces male gametes throughout postnatal life. However, experimental evaluation of SSCs in postnatal testes is complicated because these cells are extremely rare and few defining morphology or biochemical characteristics are known. In this study, we used the spermatogonial transplantation functional assay, combined with fluorescence-activated cell sorting (FACS) analysis to identify cellular, biochemical and surface antigenic characteristics of SSCs in rat testes during development. Our results demonstrated that forward scatter (FSc)^hi, side scatter (SSc)^hi, mitochondria membrane potential (ΔΨm)^lo, Ep-CAM⁺, Thy-1⁺, β3-integrin⁺ stem cells in neonate rat testes become SSc^lo, ΔΨm^hi, Ep-CAM⁺, Thy-1^lo, β3-integrin⁻ stem cells in pup rat testes. Furthermore, prospective identification of rat testis cell populations (Ep-CAM⁺), highly enriched for SSCs (1 in 13 for neonate; 1 in 8.5 for pup) enabled us to predict the Thy-1 and β3-integrin status of stem cells in neonate and pup testes, which was subsequently confirmed by transplantation analyses. Systematic characterization of SSCs enabled the production of testis cell populations highly enriched (up to 120-fold) for SSCs and will facilitate future investigations of functional and genomic characteristics.