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Background: A large-scale evaluation of mother-to-child transmission (MTCT) with dolutegravir (DTG)-based antiretroviral treatment (ART) has not been conducted previously., Setting: Botswana was the first African country to change from efavirenz (EFV)/tenofovir (TDF)/emtricitabine (FTC) to DTG/TDF/FTC first-line ART., Methods: From April 2015 to July 2018, the Early Infant Treatment Study offered HIV DNA testing at <96 hours of life. Maternal ART regimen was available for screened infants who could be linked to the separate Tsepamo surveillance study database. We evaluated characteristics of HIV-positive infants, and compared MTCT rates by ART regimen for linked infants., Results: Of 10,622 HIV-exposed infants screened, 42 (0.40%) were HIV-positive. In total, 5064 screened infants could be linked to the surveillance database, including 1235 (24.4%) exposed to DTG/TDF/FTC and 2411 (47.6%) exposed to EFV/TDF/FTC. MTCT was rare when either regimen was started before conception: 0/213 [0.00%, 95% confidence interval (CI): 0.00% to 1.72%] on DTG, 1/1497 (0.07%, 95% CI: 0.00% to 0.37%) on EFV. MTCT was similar for women starting each ART regimen in pregnancy: 8/999 (0.80%, 95% CI: 0.35% to 1.57%) for DTG and 8/883 (0.91%, 95% CI: 0.39% to 1.78%) for EFV (risk difference 0.11%, 95% CI: -0.79% to 1.06%). Most MTCT events (4/8 with DTG, 6/9 with EFV) occurred when ART was started <90 days before delivery. Infants exposed to DTG in utero had lower baseline HIV RNA compared with other HIV-infected infants., Conclusion: In utero MTCT in Botswana remains rare in the DTG era. No significant MTCT differences were observed between DTG/TDF/FTC and EFV/TDF/FTC. Risk was highest for both groups when ART was started in the third trimester.