Background: Hepatitis E virus (HEV) seroprevalence varies by age and geography. Data on HEV seroprevalence across age groups and among people living with HIV (PLWH) in South Africa is scarce. Methods: We conducted a prospective multi-site assessment of anti-HEV IgG seroprevalence on 859 South African participants enrolled at three clinical research centres including Newtown Clinical Research Centre in Johannesburg, Be Part Research in Mbekweni, Paarl, Western Cape, and Mecru Clinical Research Unit in Garankuwa, Pretoria. Participants comprised adults aged 18 -45 years (PLWH, n = 178 and HIV-negative, n = 232), and children aged 2-17 years (n = 449). Anti-HEV IgG serostatus and antibody titer were measured using a commercial ELISA kit and a WHO reference standard. Seroprevalence was assessed by site, age group, sex, and HIV status. Results: Overall anti-HEV IgG seroprevalence was 18.0% (95% CI: 15.6-20.8). Adults had the highest seroprevalence (27.3% among all adults; 29.2% among PLWH and 25.9% in HIV-negative adults), while adolescents aged 12-17 years had the lowest (6.9%), and young children aged 6-11 years and 2-5 years had 10.3% and 13.0%, respectively. Adults had significantly higher odds of seropositivity than children (aOR 2.8, 95% CI: 1.5-5.5, p = 0.002). A significant site-specific variation was also observed among healthy adults and adolescents: Newtown Clinical Research Centre (23.0% and 14.0%) and Be Part Research (34.5% and 7.3%) had higher seroprevalence compared with those from Mecru Clinical Research Unit (17.2% and 1.5%, p = 0.0499 and 0.0262, respectively). A higher mean antibody titer observed in younger children aged 2-5 years (5.06 IU/mL), compared with adults (0.88 IU/mL among PLWH and 0.68 IU/mL among HIV-negative adults), and with older children (2.02 IU/mL in those aged 6-11 years and 0.67 IU/mL in those aged 12-17 years). Conclusions: HEV seroprevalence in South Africa was highly heterogeneous, varying markedly by age group and study site. These findings highlight the need for strengthened, integrated HEV surveillance to better define transmission patterns and to inform evidence-based considerations for prevention of infection.