Background Despite the reduction of HIV mother-to-child transmission, there are concerns regarding transmission rate in the breastfeeding period. We describe the routine uptake of 6 or 10 (6/ 10) weeks, 9 months and 18 months testing, with and without tracing, in a cohort of infants who received HIV PCR testing at birth (birth PCR) (with and without point of care (POC) testing) in a peri-urban primary health care setting in Khayelitsha, South Africa. Methods In this cohort study conducted between November 2014 and February 2018, HIV-positive mothers and their HIV-exposed babies were recruited at birth and all babies were tested with birth PCR. Results of routine 6/10 weeks PCR, 9 months and 18 months testing were followed up by a patient tracer. We compared testing at 6/10 weeks with a subgroup from historical cohort who was not tested with birth PCR. Results We found that the uptake of 6/10 weeks testing was 77%, compared to 82% with tracing. When including all infants in the cascade and comparing to a historical cohort without birth testing, we found that infants who tested a birth were 22% more likely to have a 6/10 weeks test compared to those not tested at birth. There was no significant difference between the uptake of 6/10 weeks testing after birth PCR POC versus birth PCR testing without POC. Uptake of 9 months and 18 months testing was 39% and 24% respectively. With intense tracing efforts, uptake increased to 45% and 34% respectively. Conclusion Uptake of HIV testing for HIV-exposed uninfected infants in the first 18 months of life shows good completion of the 6/10 weeks PCR but suboptimal uptake of HIV testing at 9 months and 18 months, despite tracing efforts. Birth PCR testing did not negatively affect uptake of the 6/10 weeks HIV test compared to no birth PCR testing.