Rubella infection during pregnancy may cause foetal death or congenital rubella syndrome. In South Africa, the national public immunization programme does not include rubella vaccination. The aim of this study was to evaluate rubella sero-epidemiology in pregnant South African women living with and without HIV. Methods: Serum samples obtained from women living with HIV (n = 552) and without HIV (n = 552) were tested for rubella immunoglobulin G antibodies using an ELISA. The proportions of women with seronegative titres (<8 IU/ml) and seropositive titres (!11 IU/ml), and geometric mean titres (GMT) were compared by age group and HIV status. Results: The overall proportion of rubella seropositivity was 97.8%. The proportion of seropositive women increased with age group (18-25 years: 97.0%; 26-32 years: 97.7%; 33-40 years: 99.3%; p = 0.047 after adjusting for HIV status). Similar proportions of women living with and without HIV were seropositive. Conclusions: Rubella immunity was high among South African pregnant women living with and without HIV in the absence of rubella vaccination in the public immunization programme. However, a lower percentage of younger women had seropositive titres, indicating the need for routine rubella vaccination after an increase in vaccine coverage rates.