Routine screening is recommended for HIV detection. HIV risk estimation remains important. Our goal was to validate the Denver HIV Risk Score using a national cohort from the Centers for Disease Control and Prevention. Patients of 13 years and older were included, 4,830,941 HIV tests were performed, and 0.6% newly diagnosed infections were identified. Of all visits, 9% were very low risk (HIV prevalence = 0.20%), 27% low risk (HIV prevalence = 0.17%), 41% moderate risk (HIV prevalence = 0.39%), 17% high risk (HIV prevalence = 1.19%), and 6% very high risk (HIV prevalence = 3.57%). The Denver HIV Risk Score accurately categorized patients into different HIV risk groups.