AbstractObjectivesSince 2018, the World Health Organization has recommended dolutegravir (DTG)‐containing antiretroviral therapy (ART) for most people living with HIV. Country programmes across Africa have subsequently transitioned from other, mostly nonnucleoside reverse transcriptase inhibitor (NNRTI)‐based ART to DTG‐based ART. This study aims to assess the virological impact of programmatic transitioning to DTG‐based ART in Lesotho.MethodsThe prospective Dolutegravir in Real‐Life in Lesotho (DO‐REAL) cohort enrols people living with HIV initiating or transitioning to DTG‐based ART in Lesotho. Here, we present data from participants who transitioned from NNRTI‐ to DTG‐based ART between February and December 2020. Blood samples collected at transition and at 16 weeks’ follow‐up (window 8–32 weeks) were used for viral load (VL) and resistance testing.ResultsAmong 1347 participants, follow‐up data was available for 1225. The majority (60%) were female, median age at transition was 47 years [interquartile range (IQR): 38–56], and median (IQR) time since ART initiation was 5.9 (3.5–9.0) years. Among those with complete VL data, the rate of viral suppression to < 100 copies/mL was 1093/1116 (98%) before, 1073/1116 (96%) at, and 1098/1116 (98%) after transition. Even among those with a VL ≥ 100 copies/mL at transition, 42/44 (95%) achieved suppression to < 100 copies/mL at follow‐up. Seven participants had a VL ≥ 1000 copies/mL at follow‐up and did not harbour any integrase mutations associated with resistance to DTG.ConclusionsThe high levels of viral suppression observed are encouraging regarding virological outcomes upon programmatic transitioning from NNRTI‐ to DTG‐based ART.