Introduction: Pregnant women in sub-Saharan Africa have high risk of HIV acquisition, yet approaches for measuring maternal HIV incidence using routine surveillance systems are undefined. We used programmatic data from routine antenatal care (ANC) HIV testing in Botswana to measure real-world HIV incidence during pregnancy. Methods: From January 2018 to September 2019, the Botswana Ministry of Health and Wellness implemented an HIV testing program at 139 ANC clinics. The program captured information on testers’ age, testing date and result, and antiretroviral treatment (ART) initiation. In our analysis, we excluded individuals who previously tested HIV-positive prior to their first ANC visit. We defined incident HIV infection as testing HIV-positive at an ANC visit after a prior HIV-negative result within ANC. Results: Overall, 29,570 pregnant women (median age 26 years, IQR 22-31) tested for HIV at ANC clinics: 3% (836) tested HIV-positive at their first recorded ANC visit and 97% tested HIV-negative (28,734). Of those who tested HIV-negative, 28% (7,940/28,734) had a repeat HIV test recorded at ANC. The median time to HIV re-testing was 92 days (IQR 70-112). In total, 17 previously undiagnosed HIV infections were detected (HIV incidence 8 per 1,000 person-years, 95% CI 0.5-1.3). ART initiation among women newly diagnosed with HIV at ANC (853) was 88% (671/762). Conclusions: In Botswana, real-world HIV incidence among pregnant women at ANC remains above levels of HIV epidemic control (≤1 per 1,000 person-years). This study shows how HIV programmatic data can answer timely population-level epidemiological questions and inform ongoing implementation of HIV prevention and treatment programs.