Surveillance programmes for prevention of mother-to-child transmission of HIV (PMTCT) fail to quantify numbers of infant HIV infections averted, often because of poor postnatal follow-up. Additionally, infected infants are often not identified early and only gain access to comprehensive HIV care and treatment late in their disease. Anonymous, unlinked, HIV prevalence testing was conducted on dried blood spot (DBS) samples from all infants attending 6 week immunization clinics at seven primary health care clinics offering PMTCT. Samples were tested for HIV antibodies (indicating maternal HIV infection) and those determined to be from HIV-exposed infants were tested for HIV RNA by polymerase chain reaction. Infant and child mortality rates were determined using birth histories. Samples were collected from 2489 infants aged 4-8 weeks. HIV antibodies were identified in 931 infants [37.4%; 95% confidence interval (CI), 35.4-39.4], of whom 188 were HIV RNA positive. The estimated vertical transmission rate (VTR) was 20.2% (95% CI, 17.8-23.1%); 7.5% of all infants at this age were infected. Amongst mothers who reported that they had taken single-dose nevirapine for PMTCT, VTR was 15.0%. Amongst women who reported being HIV uninfected but whose infants had HIV antibodies, VTR was 30.5%. Infant mortality rates in KwaZulu Natal increased from 28/1000 live births in 1990-1994 to 92/1000 in 2000-2004. Anonymous HIV prevalence screening of all infants at immunization clinics is feasible to monitor the impact of PMTCT programmes on peripartum infection; linked screening could identify infected children early for referral into care and treatment programmes.