Background. Large cohorts of HIV-1 perinatally infected children with long-term follow-up in developing countries are limited. Objectives. To explore rates and predictors of virological failure in a paediatric cohort. Methods. A 10-year retrospective study was conducted from January 2004 to December 2013 to determine the incidence of and factors associated with virological failure among 1 659 HIV perinatally infected children in a public sector setting in South Africa (SA). Children aged <17 years who initiated first-line antiretroviral therapy between 1 January 2004 and 31 December 2013 and had at least 5 years of HIV viral load measurements were eligible. Results. The 1 659 children contributed 7 075 person-years of follow-up (PYFU). In the initial cohort of 2 024 children, 51.0% were male and 62.0% were aged <5 years. The incidence of virological failure was 18.7 per 100 PYFU. Virological failure was associated with male gender, death of the mother, concurrent tuberculosis treatment and World Health Organization stage IV disease. Of the 320 HIV isolates successfully amplified, 249 (77.8%) had drug resistance mutations. Conclusions. We observed high rates of virological failure and emergence of HIV drug resistance mutations. Despite gains made by SA in the treatment of HIV, such results challenge the country's ability to meet global targets of 90% viral suppression by 2020.