Background: HIV-related stigmatisation and discrimination by young children towards their peers have important consequences at the individual level and for our response to the epidemic, yet research on this area is limited. Methods: We used nationally representative data to examine discrimination of HIV-positive children by grade six students (n = 39,664) across nine countries in Southern Africa: Botswana, Lesotho, Malawi, Mozambique, Namibia, South Africa, Swaziland, Zambia and Zimbabwe. Descriptive statistics are used to compare discrimination by country, gender, geographic location and socioeconomic status. Multivariate logistic regression is employed to assess potential determinants of discrimination. Results: The levels and determinants of discrimination varied significantly between the nine countries. While one in ten students in Botswana, Malawi, South Africa and Swaziland would “avoid or shun” an HIV positive friend, the proportions in Lesotho, Mozambique, Zambia and Zimbabwe were twice as high (approximately 20%). A large proportion of students believed that HIV positive children should not be allowed to continue to attend school, particularly in Zambia (33%), Lesotho (37%) and Zimbabwe (42%). The corresponding figures for Malawi and Swaziland were significantly lower at 13% and 12% respectively. Small differences were found by gender. Children from rural areas and poorer schools were much more likely to discriminate than those from urban areas and wealthier schools. Importantly, we identified factors consistently associated with discrimination across the region: students with greater exposure to HIV information, better general HIV knowledge and fewer misconceptions about transmission of HIV via casual contact were less likely to report discrimination. Conclusions: Our study points toward the need for early interventions (grade six or before) to reduce stigma and discrimination among children, especially in schools situated in rural areas and poorer communities. In particular, interventions should focus on correcting misconceptions that HIV can be transmitted via casual contact.