Purpose: The Danish Registry of Diabetic Retinopathy includes information from >200 000 patients who attends diabetic retinopathy (DR) screening in Denmark. Screening of patients with uncomplicated type 2 diabetes is often performed by practicing ophthalmologists, while patients with type 1 and complicated type 2 diabetes attends screening at hospitals. We performed a clinical reliability study of retinal images from Danish screening facilities to explore the inter-grader agreement between the primary screening ophthalmologist and a blinded, certified grader. Methods: Invitations to participate were sent to screening facilities across Denmark. The primary grader uploaded fundus photographs with information on estimated level of DR (International Clinical Diabetic Retinopathy scale as 0 [no DR], 1–3 [mild, moderate or severe nonproliferative DR {NPDR}], or 4 [proliferative DR {PDR}]), region of screening, image style, and screening facility. Images were then regraded by a blinded, certified, secondary grader. Weighted kappa analysis was performed to evaluate agreement. Results: Fundus photographs from 230 patients (458 eyes) were received from practicing ophthalmologists (52.6%) and hospital-based grading centres (47.4%) from all Danish regions. Reported levels of DR by the primary graders were 66.8%, 12.2%, 13.1%, 1.3% and 5.5% for DR levels 0–4. The overall agreement between primary and secondary graders was 93% (κ = 0.83). Based on screening facility agreement was 96% (κ = 0.89) and 90% (κ = 0.76) for practicing ophthalmologists and hospital-based graders. Conclusion: In this nationwide study, we observed a high overall inter-grader agreement and based on this, it is reasonable to assume that reported DR gradings in the screening programme in Denmark, accurately reflect the truth.