Introduction PCR 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. Methods 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. Results 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 to 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). Conclusions 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 programme goals, structured interventions are required to support follow-up EID services after negative birth-test results.