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Background: Access to antiretroviral therapy (ART) for the human immunodeficiency virus type 1 (HIV-1) has lowered morbidity and mortality in people living with HIV. Unfortunately, over time, some experience the development of viral drug resistance (DR) under therapy. Little is known about the percentages of people experiencing DR, and about the genetic patterns of DR among patients failing second-line ART in Lesotho. This study aimed to assess the prevalence of HIV-1 resistance-associated mutations (RAMs) in the viral target genes of the respective drugs in individuals with unsuppressed viral loads while taking second-line ART. Methods: In a retrospective cross-sectional study, we sequenced all available plasma samples from individuals with a viral load measurement = 1 000 copies/mL while taking second-line ART between January 2016 and October 2020. Sequencing was performed using the commercial SeqStudio™ Genetic Analyser at the Seboche Mission Hospital Laboratory. Results: Out of 55 samples meeting the eligibility criteria, 30 samples were successfully amplified and sequenced. The median age of patients was 41 years (interquartile range [IQR] 30-49), and the majority (62%) of participants were female. The median duration on a second-line ART regimen at the time of phlebotomy was 1.9 years (IQR 0.5-3.0). Most participants were taking ritonavirboosted lopinavir-based ART as their second-line therapy. Major RAMs describing those mutations in targeted gene therapy reverse transcriptase (RT) and/or protease (PR) that led to virological treatment failure, were observed in 62% of participants; one patient had major RAMs in the PR region, while 18 had RAMs in the RT region. Conclusion: In our study, we found that 31% of the included individuals (10/30) had RAMs conferring resistance to their second-line regimen during the first two years after switching to second-line ART. [ABSTRACT FROM AUTHOR]