AimTo establish cut‐off values for anti‐Müllerian hormone (AMH) and antral follicle count (AFC) in the diagnostic criteria for polycystic ovary syndrome (PCOS) applicable to the Japan Society of Obstetrics and Gynecology (JSOG) 2024 criteria and the Rotterdam/International Evidence‐Based Guideline for the assessment and management of PCOS (IEBG) 2023 criteria based on a nationwide survey, respectively, taking into account age, assays, and structure of the diagnostic criteria.MethodsData were collected for 986 PCOS cases and 965 control cases using a national survey in Japan and used to establish cut‐off values for AMH and AFC.ResultsSerum AMH levels were significantly higher in the PCOS group compared to the control group. Serum AMH showed a significant negative correlation with age and significant positive correlation with AFC in both groups. In multiple regression analysis, serum AMH level was independently affected by AFC and total testosterone. AMH cut‐off values suitable for the JSOG 2024 criteria and the Rotterdam/IEBG 2023 criteria were separately established for the 20–29 and 30–39 years of age groups, respectively, and for Access, Lumipulse and Elecsys/ECLusys, respectively. AFC cut‐off values suitable for the JSOG 2024 criteria and Rotterdam/IEBG 2023 criteria were also established separately. AFC exhibited statistically greater variability than AMH.ConclusionThe serum AMH level is the biochemical representation of ovarian findings in PCOS and considered objective and highly reliable. Therefore, it could serve as a surrogate for AFC as a marker of polycystic ovarian morphology in diagnostic criteria.