OBJECTIVE: To assess the cost effectiveness of universal repeat screening for human immunodeficiency virus (HIV) infection in the third trimester of pregnancy. METHODS: A decision-analytic model was constructed to compare two strategies: screening for HIV infection in the first trimester alone compared with the addition of repeat screening in the third trimester. Probabilities, costs, and utilities were derived from the literature and varied in sensitivity analyses. The assumed incidence of HIV infection in pregnancy was 0.0145% or 14.5 per 100,000. Outcomes included costs (in 2022 U.S. dollars), maternal and neonatal quality-adjusted life-years (QALYs), and cases of neonatal HIV infection. Our theoretical cohort contained 3.8 million pregnant individuals, the approximate number of births per year in the United States. The willingness-to-pay threshold was set at $100,000/QALY. We performed univariable and multivariable sensitivity analyses to determine inputs that most influenced the model. RESULTS: Universal third-trimester screening prevented 133 cases of neonatal HIV infection in this theoretical cohort. Universal third-trimester screening led to an increased cost of $17.54 million and 2,732 increased QALYs, with an incremental cost-effectiveness ratio of $6,418.56 per QALY, less than the willingness-to-pay threshold. In a univariate sensitivity analysis, third-trimester screening remained cost effective with variation of HIV incidence in pregnancy to as low as 0.0052%. CONCLUSION: In a theoretical U.S.-based cohort of pregnant individuals, universal repeat screening for HIV infection in the third trimester was found to be cost effective and to reduce vertical transmission of HIV. These results merit consideration of a broader HIV-screening program in the third trimester.