Significantly improved survival rates in children and adolescents with cancer have put fertility preservation high on the pediatric oncology agenda. Here we report a retrospective single-center study of 13 years experience of ovarian tissue cryopreservation (OTC) before sterilizing treatment in order to define the safety/benefits of OTC and study clinical/hormonal outcomes in girls. From 2000 to 2013, OTC was performed in 36 girls: eight had non-malignant disease and 28 had malignant disease. Laparoscopy was used to collect a third of each ovary that was frozen by a slow cooling protocol. Indications for OTC were 13 auto-, 19 allo-stem-cell-transplantation and 4 sterilizing chemotherapy. Ovarian tissue harvested by intraumbilical laparoscopy led to no major postoperative complications and did not delay chemotherapy. Histological analysis of ovarian tissue showed an average of 9 primordial follicles/mm(2) [0-83] and no malignant cells were identified. Median post-harvest follow-up was 36 months [1-112]: 26 girls were alive in complete remission and 10 had died. Hormonal results were evaluable for 27 patients (median age 17 yrs [5-26]): 16 patients were in premature ovarian insufficiency. OTC sampling one third of each ovary appears to be an appropriate approach to preserve fertility in children without consequences on subsequent therapeutic program.