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To determine the prevalence and risk factors of retinopathy and validity of the current diagnostic cut-offs for diabetes by using data of health check-up examinees.The study comprises 1,864 Japanese who participated in the general health check-up program and did not have a previous history of cardiovascular disease. Non-mydriatic 45° digital fundus photographs were taken twice annually. Multivariate logistic regression model was used to identify risk factors for retinopathy in participants without previously diagnosed diabetes.The overall prevalence of retinopathy in participants with and without previously diagnosed diabetes were 23.3% (28/120) and 4.2% (74/1,744), respectively. Univariate logistic regression analysis identified age, systolic blood pressure (SBP), fasting plasma glucose (FPG) and hemoglobin A1c (HbA1c) as risk factors for retinopathy. Multivariate logistic regression analysis showed that FPG or both HbA1c and SBP were significant, positive and independent risk factors for retinopathy. The prevalence of retinopathy increased with deterioration of glucose categories (P < 0.001 for FPG or HbA1c). However, a statistically significant increased risk of retinopathy remained only in participants with FPG ≥ 7.0 mmol/L or HbA1c ≥ 6.5% compared with those with the lowest quartile of glucose in the participants without previously diagnosed diabetes after adjusting for age and SBP.The prevalence of retinopathy was 4.2%, and FPG or both HbA1c and SBP were positive and independent risk factors for retinopathy in health check-up examinees without previously diagnosed diabetes. The FPG 7.0 mmol/L or HbA1c 6.5% seems to be appropriate to diagnose diabetes in view of its association with retinopathy.