PURPOSE: Many individuals with differences of sex development experience subfertility. This study describes a novel gonadal tissue cryopreservation protocol for those individuals. MATERIALS AND METHODS: Before 2018, patients with differences of sex development electing gonadal tissue cryopreservation were enrolled in a cancer-focused protocol. Thereafter, our hospital developed a protocol specifically for these patients undergoing gonadectomy due to neoplasia risk. Protocol development steps and procedures are reported. A retrospective chart review was conducted for patient characteristics and cryopreservation choices. RESULTS: During the institutional review board approval process, there were multidisciplinary meetings with stakeholders. The main concerns discussed included preoperative counseling, pathological evaluation and final tissue disposition. Detailed multidisciplinary preoperative counseling is provided regarding potential gonadal tissue cryopreservation for patients undergoing gonadectomy. For enrolled patients, the gonad is bisected after removal. Half is sent to pathology, and half processed for cryopreservation. If neoplasia is noted, cryopreserved tissue is recalled for further pathologic analysis. Postoperative counseling is performed after pathology is available, and the final gonadal tissue cryopreservation decision is made. During the study period, seven patients elected to attempt gonadal tissue cryopreservation: five diagnoses, median age 10.99 years [IQR 1.29-14.84], 4/7 [57%] with germ cells, all of whom stored tissue. CONCLUSIONS: Gonadal tissue cryopreservation at the time of gonadectomy is feasible for patients with differences of sex development at risk for gonadal neoplasia. The protocol described represents a template for institutions who wish to offer gonadal tissue cryopreservation to patients electing gonadectomy. Over half of patients thus far have cryopreserved gonadal tissue.