OBJECTIVE: Primary ovarian insufficiency (POI) with a 46,XX karyotype is rare in pediatric and adolescent populations. It is a heterogeneous condition with important implications for pubertal development, fertility potential, genetic counseling, and long-term health. This study aimed to characterize the clinical, genetic, and etiological spectrum of pediatric and adolescent POI and to evaluate the diagnostic contribution of genetic testing. METHODS: We retrospectively analyzed pediatric and adolescent patients diagnosed with POI before 18 years of age. Demographic characteristics, presenting features, auxological data, hormonal profiles, imaging findings, molecular investigations, treatment-related exposures, and etiological classifications were analyzed. RESULTS: Twenty-five patients were included. The median age at diagnosis was 14.83 years (interquartile range [IQR]: 11.81-15.71). Primary amenorrhea was the most common presentation, observed in 44% of patients, followed by diagnosis during endocrine surveillance for chronic diseases in 24%, short stature in 20%, and secondary amenorrhea in 12%. Genetic etiologies were identified in 56% of patients, with variants detected in MCM9, HAX1, TALDO1, HFM1, and MTRFR (C12orf65). Familial clustering was particularly prominent among patients with MCM9 and HAX1 variants. Iatrogenic POI occurred in patients previously exposed to gonadotoxic chemotherapy, radiotherapy, or gonadotoxic conditioning regimens administered before hematopoietic stem cell transplantation (HSCT). CONCLUSIONS: In this study, we present the clinical characteristics of pediatric and adolescent patients with POI, including different etiologies and phenotypic presentations. Our findings underscore the value of targeted Next-Generation Sequencing (NGS) analysis in routine clinical practice. Moreover, the recurrent identification of the same MCM9 variant in unrelated families raises the possibility of a founder effect.