BACKGROUND: The frequency of new HIV infections among female sex workers in sub-Saharan Africa is poorly understood. We used routinely collected data that enable unique identification of repeat HIV testers to assess temporal trends in seroconversion and identify associated risk factors for female sex workers accessing Sisters with a Voice, Zimbabwe's national sex worker programme. METHODS: We pooled HIV testing data gathered between Sept 15, 2009, and Dec 31, 2019, from 36 Sisters programme sites in Zimbabwe. We included female sex workers aged 16 years or older with an HIV-negative test and at least one subsequent programme test. We calculated HIV seroconversion rates (using the midpoint between the HIV-positive test and the last negative test as the seroconversion date) and estimated rate ratios to compare 2-year periods by using Poisson regression, with robust SEs to account for clustering by site and adjusting for age and testing frequency to assess temporal trends. We did sensitivity analyses to explore assumptions about seroconversion dates and the effects of variation in follow-up time on our conclusions. FINDINGS: Our analysis included data for 6665 female sex workers, 441 (7%) of whom seroconverted. The overall seroconversion rate was 3·8 (95% CI 3·4-4·2) per 100 person-years at risk. Seroconversion rates fell with time since first negative HIV test. After adjustment, there was evidence of a decrease in seroconversion rates from 2009 to 2019 (p=0·0053). In adjusted analyses, being younger than 25 years, and having a sexually transmitted infection diagnosis at a previous visit, were significantly associated with increased seroconversion rates. Our findings were mostly robust to sensitivity analyses, but when 1 month before an HIV-positive test was used as the seroconversion date, seroconversion rates no longer fell with time. INTERPRETATION: We identified high rates of seroconversion shortly after linkage to programme services, which emphasises the need to strengthen HIV prevention programmes from first contact with female sex workers in Zimbabwe. New infections among female sex workers remain challenging to measure, but longitudinal analysis of routine testing data can provide valuable insights into seroconversion rates and associated risk factors. FUNDING: UN Population Fund, Deutsche Gesellschaft für Internationale Zusammenarbeit, the Bill & Melinda Gates Foundation, The Global Fund to Fight AIDS, Tuberculosis and Malaria, US President's Emergency Plan for AIDS Relief, US Agency for International Development, and the Elton John AIDS Foundation.