Since the 1990s, perinatal transmission of HIV has decreased substantially, largely as a result of improved detection secondary to routine HIV screening in pregnancy and the use of antiretroviral therapy. However, despite reductions in HIV transmission, elimination of perinatal transmission, defined as an incidence of perinatal HIV infection of <1 per 100,000 live births and a transmission rate of <1%, remains elusive. An estimated 80% of perinatal transmissions occur after 36 weeks’ gestation, which highlights the importance of diagnosis and treatment of maternal HIV infection before the highest-risk period for perinatal transmission. With timely identification of seroconversion, intrapartum and neonatal interventions can lower the risk of perinatal transmission from 25% to 10%, substantially reducing perinatal transmission events. The American College of Obstetricians and Gynecologists and the Centers for Disease Control and Prevention recommend that routine HIV testing be performed in all pregnancies, as early in the prenatal course as possible. Third-trimester repeat testing is only recommended for individuals known to be at high risk of acquiring HIV (ie, those who are incarcerated; who reside in jurisdictions with elevated HIV incidence; who are receiving care in facilities that have an HIV incidence in pregnant women > 1 per 1000 per year; or have signs or symptoms of acute HIV). However, among reproductive-age women, heterosexual intercourse is the most common mode of HIV transmission, and the risk of HIV seroconversion is greater during pregnancy than outside of pregnancy. Furthermore, state statutes for HIV testing in pregnancy are largely lacking. In this clinical opinion, we reviewed the evidence in support of universal third-trimester repeat HIV testing in pregnancy using a successful state-mandated testing program in Illinois. In addition, we provided clinical recommendations to further reduce missed perinatal transmission cases by implementing universal third-trimester repeat testing, obtaining hospital buy-in, monitoring testing adherence, bridging communications across multidisciplinary teams, and engaging clinicians in advocacy work.