South Africa has experienced declining marriage rates and the increasing practice of cohabitation without marriage. This study aims to improve the understanding of the relationship between marital status and HIV in South Africa, an HIV hyperendemic country, through an analysis of findings from the 2012 South African National HIV Prevalence, Incidence and Behaviour Survey. The nationally representative population-based cross-sectional survey collected data on HIV and socio-demographic and behavioural determinants in South Africa. This analysis considered respondents aged 16 years and older who consented to participate in the survey and provided dried blood spot specimens for HIV testing (N = 17,356). After controlling for age, race, having multiple sexual partners, condom use at last sex, urban/rural dwelling and level of household income, those who were married living with their spouse had significantly reduced odds of being HIV-positive compared to all other marital spouses groups. HIV incidence was 0.27% among respondents who were married living with their spouses; the highest HIV incidence was found in the cohabiting group (2.91%). Later marriage (after age 24) was associated with increased odds of HIV prevalence. Our analysis suggests an association between marital status and HIV prevalence and incidence in contemporary South Africa, where odds of being HIV-positive were found to be lower among married individuals who lived with their spouses compared to all other marital status groups. HIV prevention messages therefore need to be targeted to unmarried populations, especially cohabitating populations. As low socio-economic status, low social cohesion and the resulting destabilization of sexual relationships may explain the increased risk of HIV among unmarried populations, it is necessary to address structural issues including poverty that create an environment unfavourable to stable sexual relationships.