OBJECTIVE: Many cancer survivors may experience irreversible infertility due to chemotherapy treatment for childhood cancer. In this study, spermatogenesis development was evaluated following the grafting of fresh and frozen-thawed testicular tissue from neonatal mice to the epididymal fat of adult mice. METHODS: After bilateral castration of recipient mice, fresh or frozen-thawed neonatal testis tissues were grafted into the epididymal fat of the mice. Grafted testicular tissue was evaluated eight weeks after implantation using H&E staining, real-time PCR, immunofluorescence staining, and TUNEL assay. Blood was drawn from recipient mice to determine testosterone, FSH, and LH levels. RESULTS: A gradient of different types of germ cells, from spermatogonia to elongated spermatids was observed. The upregulation of meiotic and post-meiotic genes and proteins in fresh and frozen grafted groups confirmed the progression of meiosis and post-meiosis in grafted tissues. There were no significant differences in the expression of apoptosis and necrosis genes between the grafted and non-grafted control groups. Additionally, no significant differences were observed between the control and experimental groups in hormonal assessments. CONCLUSIONS: The optimal hormonal and temperature conditions of the epididymal fat could support spermatogenesis in grafted immature testicular tissue. This grafting technique could pave the way for fertility preservation.