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Polymerase chain reaction testing at birth (“birth-testing”) is suggested by new World Health Organization guidelines for rapid diagnosis of infants infected with HIV in utero. However, there are few data on the implementation of this approach in sub-Saharan Africa, and whether birth testing affects uptake of subsequent routine early infant diagnosis (EID) testing at 6–10 weeks of age is unknown.We reviewed 575 consecutive infants undergoing targeted high-risk birth testing in Cape Town, South Africa, and matched those testing HIV negative at birth (n = 551) to HIV-exposed infants who did not receive birth testing (n = 551). Maternal and infant clinical and demographic data, including EID testing uptake, were abstracted from routine records.Overall, 3.8% of all birth tests conducted were positive while later EID testing positivity rates were 0.5% for those infants testing HIV negative at birth and 0.4% for those without birth testing. Infants who underwent birth testing were less likely to present for later EID compared with those without a birth test (73% vs. 85%; P < 0.001). This difference persisted after adjusting for maternal and infant characteristics (adjusted odds ratio, 0.60; 95% confidence interval: 0.41–0.86) and across demographic and clinical subgroups. Infants undergoing birth testing also presented for later EID at a significantly older age (mean age, 60 vs. 50 days; P < 0.001).While the yield of targeted high-risk birth testing in this setting appears high, neonates testing HIV negative at birth may be less likely to present for subsequent EID testing. For birth testing implementation to contribute to overall EID program goals, structured interventions are required to support follow-up EID services after negative birth test results.