Treatment of cancer in children is increasingly successful but leaves many prepubertal boys suffering from infertility or subfertility later in life. A current strategy to preserve fertility in these boys is to cryopreserve a testicular biopsy prior to treatment with the expectation of future technologies allowing for the reintroduction of stem cells and restoration of spermatogenesis. Spermatogonial stem cells (SSCs) form the basis of male reproduction, differentiating into all germ cell types, including mature spermatozoa and can regenerate spermatogenesis following transplantation into an infertile testis. Here, we demonstrate that rat SSCs frozen for more than 20 years can be transplanted into recipient mice and produce all differentiating germ cell types. However, compared with freshly isolated cells or those frozen for a short period of time, long-frozen cells do not colonize efficiently and showed reduced production of spermatids. Single-cell RNA sequencing revealed similar profiles of gene expression changes between short- and long-frozen cells as compared with fresh immediately after thawing. Conversely, following transplantation, long-frozen samples showed enhanced stem cell signaling in the undifferentiated spermatogonia compartment, consistent with self-renewal and a lack of differentiation. In addition, long-frozen samples showed fewer round spermatids with detectable protamine expression, suggesting a partial block of spermatogenesis after meiosis resulting in a lack of elongating spermatids. These findings strongly suggest that prolonged cryopreservation can impact the success of transplantation to produce spermatogenesis, which may not be revealed by analysis of the cells immediately after thawing. Our analysis uncovered persistent effects of long-term freezing not found in other cryopreservation studies that lacked functional regeneration of the tissue and this phenomenon must be accounted for any future therapeutic application.