To investigate whether mutations in the minichromosome maintenance complex component 8 (MCM8) gene were present in 192 patients with sporadic primary ovarian insufficiency (POI).Retrospective case-control cohort study.University-based reproductive medicine center.A total of 192 patients with sporadic POI and 312 control women with regular menstruation (192 age-matched women and 120 women >45 years old).Sanger sequencing was performed in patients with sporadic POI, and potentially pathogenic variants were confirmed in matched controls. DNA damage was induced by mitomycinC (MMC) treatment, and DNA repair capacity was evaluated by histone H2AX phosphorylation level.Sanger sequencing for MCM8 was performed in 192 patients with sporadic POI, and functional experiments were performed to explore the deleterious effects of mutations identified.Two novel missense variants in MCM8, c. A950T (p. H317L), and c. A1802G (p. H601R), were identified in two patients with POI but absent in 312 controls (the upper 90% confidence limit for the proportion 2/192 is 2.24%). The HeLa cells overexpressing mutant p. H317L and p. H601R showed higher sensitivity to MMC compared with wild type. Furthermore, mutant p. H317L showed decreased repair capacity after MMC treatment with much more histone H2AX phosphorylation remaining after 2 hours of recovery.Our result suggests novel mutations p. H317L and p. H601R in the MCM8 gene are potentially causative for POI by dysfunctional DNA repair.