The selective breeding of farmed species is an essential task for sustainable aquaculture. Barramundi (Asian seabass: Lates calcarifer) is no exception in this respect, and artificial breeding is underway to establish strains with valuable genetic traits. For the long-term preservation of the many strains carrying diverse genetic characteristics, producing offspring derived from cryopreserved germ cells by transplantation into recipients is a powerful tool. Therefore, we aimed to develop a germ cell transplantation method, an essential step in obtaining the next generation derived from frozen germ cells in this study. First, testicular sections of various-sized fish (75 g–8 kg) were observed to identify the suitable maturation stages of donors. Immunocytochemistry with the antibody specifically recognizing the cell surface antigen of type A spermatogonia (ASGs) was performed. As a result, fish with <500 g of body weight possessed a higher number of ASGs, and their frequency of occurrence was also higher in their gonads. Although it was difficult to obtain sufficient amounts of testicular cell suspensions used for transplantation with conventional enzyme digestion, pretreatment using proteinase K was quite effective in dissociating the testicular tissues of this species. Next, 4–20 days post hatching (dph) larvae were sampled in a time-course manner. The development of their genital ridges was observed to identify the suitable developmental stages of larvae as recipients. By 16 dph, endogenous primordial germ cells were incorporated into the genital ridges and covered by somatic cell layers. This suggests that 16-dph or younger larvae are optimal for use as recipients. Lastly, we transplanted testicular cells, prepared using the above-mentioned optimal conditions, into the peritoneal cavity of 10–14 dph larvae. The transplantation success rate was compared for each total length of the recipient larvae. As a result, the transplantation success rates of 3.21 ± 0.093 mm, 4.00 ± 0.045 mm, and 5.01 ± 0.13 mm total length recipients were 77.2% ± 12.3%, 57.5% ± 4.7%, and 45.3% ± 4.9%, respectively. It was indicated by these results that donors weighing <500 g and larvae with a total length of about 3.2 mm are suitable for germ cell transplantation in barramundi. © 2023 Elsevier B.V.