In South Africa, adolescents constitute a key population at high risk of HIV acquisition. However, little is known about HIV transmission among students within schools. This study was undertaken to assess the risk factors for HIV infection and the extent of transmission among rural high school students. Between February and May 2012, consenting students from five randomly selected public sector high schools in rural KwaZulu-Natal participated in an anonymous cross-sectional survey. Dried blood spot samples were collected and tested for HIV. β-Human chorionic gonadotropin (βHCG) levels were measured in females for pregnancy. Family circumstances as well as sociodemographic and behavioral factors were assessed as potential risk factors. A subset (106/148, 72%) of HIV-positive samples underwent gag p17p24 sequencing for phylogenetic analysis. A total of 3,242 students (81.7% of enrolled students) participated. HIV prevalence was 6.8% [95% confidence interval (CI) 3.9-9.8%] in girls and 2.7% (CI 1.6-3.8%) in boys [adjusted odds ratio (aOR)=3.0, CI 2.4-3.8; p<0.001]. HIV prevalence increased from 4.6% (95% CI 1.9-7.3) in the 12- to 15-year-old girls to 23.1% (95% CI 7.7-38.5) in girls over 20 years, while in boys HIV prevalence increased from 2.7% (95% CI 0.6-4.9) in the 12- to15-year-old boys to 11.1% (95% CI 2.7-19.4) in those over 20 years. Sequencing of samples obtained from students revealed only two clusters, suggesting within-school transmission and three interschool clusters, while the remainder was most likely acquired from sources other than those currently found in students attending the school concerned. HIV prevalence in both girls (aOR=3.6, CI 2.9-4.5; p<0.001) and boys (aOR=2.8, CI 1.2-6.2; p=0.01) was higher in those without a living biological mother. The high burden of HIV infection among students was not associated with intraschool transmission in this rural setting. Lack of a living parent is an important factor defining high risk in this group of adolescents.