Background: Ovarian insufficiency (OI) and infertility are common and devastating late effects of cancer treatment and hematopoietic stem cell transplantation (HSCT). In children, gonadal insufficiency may subsequently lead to abnormal pubertal development. The aim of this study was to assess the cumulative incidence of OI and the need for hormonal induction of pubertal development after HSCT in childhood. We additionally assessed HSCT-related risk factors for OI. Procedures: A single center cohort study was undertaken of female patients transplanted during childhood, surviving at least 2 years post-HSCT and who were at least 10 years old at initiation of the study. Of 141 eligible patients, 109 were included and hormone levels and clinical data of these patients during follow-up were collected. Risk factors for OI were analyzed by multivariate Cox regression analysis. Results: Cumulative incidence of OI was 56% at a median follow-up of 7.2 years. Eight patients, initially diagnosed with OI, showed recovery of ovarian function over time. Hormonal induction of puberty was necessary in 44% of females who were pre-pubertal or pubertal at HSCT. In multivariate analysis, more advanced pubertal stage at HSCT was associated with OI. We found a trend for an association of busulfan with OI in patients conditioned with chemotherapy only. Conclusions: The incidence of OI after HSCT was high and associated with more advanced pubertal stage at HSCT. Almost half of the females who were pre-pubertal or pubertal at HSCT required hormonal induction of pubertal development.