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The World Health Organization recommends that all blood donations be screened for transfusion transmissible infections; these data are currently not incorporated into national disease surveillance efforts. We set out to use routinely collected data from blood donors in Blantyre district, Malawi to explore HIV and syphilis prevalence and identify sero-conversions among repeat donors. We conducted a retrospective cohort analysis of blood donation data collected by the Malawi Blood Transfusion Service from 2015 to 2021. All blood donations were routinely screened for HIV and syphilis. We characterized donor demographics as well as screening outcomes, including identifying sero-conversions among repeat donors who previously tested negative on their last donation. A total of 23,280 donations from 5,051 donors were recorded, with a median frequency of donations of 3 (IQR:2-6). Most donors were male (4,294; 85%) and students (3,262; 64.6%). Prevalence of HIV at first donation was 1.0% (52/5,051) and prevalence of syphilis was 1.6% (80/5,051); 52 HIV sero-conversions and 126 syphilis sero-conversions were identified, indicating an incidence rate per 1,000 person-years of 5.9 (95% CI: 4.7, 7.4) and 13.3 (95% CI:11.4, 15.4) respectively. Students had a lower prevalence of HIV and syphilis but higher risk of syphilis seroconversion. While blood donors are generally considered a low-risk population for HIV and syphilis, we were able to identify relatively high rates of undiagnosed HIV and syphilis infections among donors. Routinely collected data from national blood donation services may be used to better understand local HIV and syphilis epidemiology, with the potential to enhance disease surveillance systems. These findings may be used to identify priority prevention areas and populations in Blantyre district that can inform targeted interventions for improved disease prevention, testing and treatment., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Singogo et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)