Background: South Africa (SA) has the highest burden of HIV infection in the world. Even though HIV testing is mandated in all hospital-based facilities in SA, it is rarely implemented in the emergency department (ED). EDs are episodic care centers that provide care to large volumes of undifferentiated patients for short periods of time, and may treat undiagnosed HIV-infected patients not captured through standard clinic based screenings. Methods and Findings: In this prospective study, we implemented the National South African HIV testing guidelines, including 24-hours a day counselor initiated HIV Counseling and Testing (HCT), at Frere Hospital in the Eastern Cape from September 1 to November 30, 2016. All patients that presented for care in the ED during the study period, and who were clinically stable and fully conscious, were eligible to be approached by HCT staff to receive a rapid point-of-care HIV test. A total of 2355 of the 9583 (24.6%) patients that presented to the ED for care during the study period were approached by the HCT staff, of whom 1852 were enrolled in the study. There was high uptake of HIV testing (78.6%) among a predominantly male (58%) patient group that mostly presented with traumatic injuries (70.8%). Four hundred (21.6%) of the enrolled patients were HIV positive, including 115 (6.2%) with previously undiagnosed HIV infection. The overall prevalence of HIV infection in females (29.8%) was twice that compared to males (15.4%), despite the burden of undiagnosed infection being similar (6.0% for all females and 6.4% for all males). Conclusions: Overall there was a high HIV testing uptake that revealed a significant burden of undiagnosed HIV infection in this setting, especially in young males. Future research should focus on testing optimization, and efficient linkage to care from the ED for antiretroviral therapy initiation