Premature ovarian insufficiency (POI) is a condition characterized by hypergonadotropic hypogonadism and amenorrhea, which rarely manifests in adolescence. Up to 50% of POI cases are idiopathic. We conducted molecular diagnostic testing of 39 patients who experienced the onset of POI before the age of 18, all of whom had a normal female karyotype. This involved FMR1 CGG repeats testing and whole-exome sequencing. None of the patients were found to have a premutation in the FMR1 gene. In 17 out of 39 patients (43.5%), variants were identified across 12 genes. A diagnosis of monogenic POI was established in 7 patients (18%) based on the detection of pathogenic and likely pathogenic variants. Syndromic forms of POI were observed in 3 cases (8%), while isolated POI was noted in 4 cases (10%). In 4 patients (10%), variants of uncertain clinical significance were found in the STAG3, LMNA, SPIDR, and NOBOX genes. Additionally, 6 patients (15.5%) had heterozygous variants on one allele in genes associated with autosomal recessive POI. We recommend that patients with onset of POI in adolescence undergo genetic counseling and molecular genetic testing.