Background For women living with HIV (WLHIV), co-infection with herpes simplex virus type 2 (HSV-2) causes severe genital ulcers and presents additional challenges for their HIV care. To inform preventive strategies, we aimed to determine the incidence and risk factors of HSV-2 positivity in a prospective cohort of South African women. Methods The CAPRISA 002 study enrolled women at acute HIV infection between 2004 and 2020. HSV-2 testing was conducted by multiplex polymerase chain reaction (PCR) assay on collected vaginal swabs up to twice annually during follow-up. We calculated incidence as the number of new cases per 100 person-years (PYs) and used Cox-proportional-hazard regression to identify factors associated with time-to-HSV-2 PCR positivity. Results At enrolment, the median age of 171 women was 24 years, interquartile range (IQR 21–28), and the estimated median days since HIV infection was 42 (IQR 22–65). Of participants tested at enrolment, HSV-2 antibody prevalence was 81.4% (105/129), and 10.6% (12/113) were positive by PCR. Among 147 women with a prior negative HSV-2 PCR diagnosis, we observed 47 new HSV-2 PCR positive cases over 424.4 PYs of follow-up, yielding an incidence rate of 11.1 cases per-100-PYs. HSV-2 PCR positivity incidence was higher among younger women (<25 years: adjusted Hazard Ratio [aHR] = 5.91, 95%CI 3.02–11.6), those with bacterial vaginosis (BV) (Nugent score 7–10: aHR = 2.17, 95%CI 1.15–4.10) and lower CD4 counts (<500 cells/μl: aHR = 2.04, 95%CI 1.08–3.87). Conclusion After acute HIV infection in women, the incidence of HSV-2 PCR positivity was associated with younger age, BV diagnosis and lower CD4 count.