Introduction Primary ovarian insufficiency (POI) affects 1% of women under 40 years old. POI is idiopathic in more than 70% of cases. Though many candidate genes have been identified in recent years, the prevalence and pathogenicity of abnormalities are still difficult to establish. Objectives Our primary objective was to evaluate the prevalence of gene variations in a large prospective multicentric POI cohort. Our secondary objective was to evaluate the correlation between phenotype and genotype. Patients and Methods Two hundred and sixty-nine well-phenotyped POI patients were screened for variants of 18 known POI genes (BMP15, DMC1, EIF2S2, FIGLA, FOXL2, FSHR, GDF9, GPR3, HFM1, LHX8, MSH5, NOBOX, NR5A1, PGRMC1, STAG3, XPNPEP2, BHLB and FSHB) by next generation sequencing (NGS). Abnormalities were classified as “variant” or “variant of unknown signification” (VUS) according to available functional tests or algorithms (SIFT, Polyphen-2, MutationTaster). Results One hundred and two patients (38%) were identified as having at least 1 genetic abnormality. Sixty-seven patients (25%) presented at least 1 variant. Forty eight patients presented at least 1 VUS (18%). Thirteen patients (5%) had combined abnormalities. NOBOX variants were the most common gene variants involved in POI (9%). Interestingly, we saw no significant differences in the previous family history of POI, ethnic origin, age at onset of POI, primary amenorrhea or secondary menstrual disturbances between the different genotypes. Conclusion In our study, a high percentage of patients presented gene variants detected by NGS analysis (38%). Every POI patient should undergo NGS analysis to improve medical cares of the patients.