Cryopreservation of testicular tissue would provide a useful tool to maintain spermatogonia, a precursor of gametes, for fish regeneration by germ cell transplantation. This study aimed to develop a cryopreservation method for spermatogonia by freezing the testicular tissue of striped catfish (Pangasianodon hypophthalmus) and testing whether cryopreserved spermatogonia exhibit transplantability. Freezing cryomedium containing L-15 medium and 1.3 M DMSO yielded the highest spermatogonia recovery rate after thawing. Supplementation with glucose and non-permeating cryoprotectants was not necessary to improve the recovery rate. We determined that an optimised equilibration time for cryopreserved testes in cryomedium included 60 min of equilibrium on ice before slow freezing (?1 °C/min), and subsequent thawing at 10 °C for 4–8 min. An Annexin V affinity assay and flow cytometry analysis were performed to identify apoptosis in cryopreserved testicular cells. Our results showed that cryoinjury of spermatogonia during cryopreservation could lead to apoptosis. Additionally, to determine the characteristics of cryopreserved spermatogonia at the molecular level, RT-qPCR was performed to quantify the expression of cxcr4b, dazl, dnd1, dnd2, nanos1, nanos2, and vasa. The results showed that compared to fresh testicular cells, frozen cells exhibited lower expression levels of cxcr4b and dazl. However, there were no significant differences in dnd1, dnd2, nanos1, and nanos2 expression levels. Although the expression level of vasa was lower in cryopreserved testes, both fresh and cryopreserved testicular cells showed positive vasa expression using in situ hybridisation. Furthermore, cryopreserved spermatogonia were transplanted into allogeniec recipient larvae and compared with fresh spermatogonia. Although the incorporation rate was lower than that of fresh spermatogonia, cryopreserved spermatogonia could be incorporated into the genital ridge of recipient larvae, demonstrating the transplantability of cryopreserved spermatogonia. Taken together, a method for cryopreservation of testicular tissue of striped catfish was optimised, and the cryopreserved testicular cells retained several spermatogenic stem cell characteristics, as well as transplantability. © 2023 Elsevier B.V.