In the Netherlands a substantial proportion of newly diagnosed human immunodeficiency virus (HIV) patients present late for care and an estimated 12-34% of people living with HIV are undiagnosed. Linkage to care of these patients is important to decrease HIV transmission and to improve individual patient outcomes. We investigated if non-targeted HIV testing in emergency departments is a useful and cost-effective way to identify these patients.In a cross-sectional multicentre study, eligible adult patients who underwent phlebotomy were given an active choice to be additionally tested for HIV. In a subset of patients, risk factors for HIV infection were asked for. A cost-effectiveness analysis was conducted.Of 7577 eligible patients, 3223 patients were tested, and two new HIV infections were diagnosed (0.06%). Both patients had risk factors for HIV infection. Non-targeted HIV testing in the emergency department was not considered cost-effective, with a cost per quality adjusted life years gained of € 77,050, more than triple the Dutch cost-effectiveness threshold of € 20,000.Non-targeted HIV testing in emergency departments in the Netherlands had a low yield of newly diagnosed HIV infections and was not cost-effective. Our data suggest that targeted HIV testing may offer an alternative approach to decrease the number of undiagnosed people living with HIV.