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Background: Children who are exposed to HIV in utero but are uninfected (HIV-exposed uninfected or HEU) are at higher risk of neurodevelopmental delays compared to children born to persons without HIV. Magnetic resonance imaging (MRI) studies have revealed differences in grey matter volumes, cerebral perfusion, and white matter changes in these children. However, MRI is costly and not widely available in areas with high HIV prevalence, like Botswana, where more than 15% of children are HEU. To address this, we explored the use of brain ultrasound, conducted by trained study nurses, as a safe, less costly, and accurate alternative method for assessing differences relating to HIV exposure status in the brain structures of neonates. Methods: Brain ultrasounds of newborns in the Following Longitudinal Outcomes to Understand, Report, Intervene and Sustain Health for Infants, Children, Adolescents who are HIV Exposed Uninfected (FLOURISH) observational study—comprising 35 HEU newborns and 24 HIV-unexposed (HU) newborns—were performed by study nurses and evaluated by a pediatric radiologist for quality and structural abnormalities, such as calcifications, cysts, and hemorrhages. Two radiologists measured extra-axial cerebrospinal fluid spaces, ventricles, and the corpus callosum. Results: Ultrasound studies of 59 newborns (59% boys; median gestational age 38.4 weeks) were completed. All studies were of diagnostic quality, with 90.2% rated as being of good or excellent quality. Structural abnormalities were rare (10.2% incidence) and did not differ by HIV exposure group. Corpus callosum length was shorter in HEU infants compared to HU infants (45.7 mm vs. 47.3 mm; p = 0.03). Other ventricular and corpus callosum measurements showed no significant variations. Conclusions: Brain ultrasounds conducted by study nurses are feasible and reveal differences in corpus callosum length between HEU and HU infants. The benefits of easier training, lower cost, and rapid deployment make ultrasound a promising screening tool in resource-limited settings.