(1) Background: We aimed to determine the prevalence of hepatitis B virus (HBV) resistance-associated mutations (RAMs) in people with HBV and human immunodeficiency virus (HBV/HIV) in Botswana. (2) Methods: We sequenced HBV deoxyribonucleic acid (DNA) from participants with HBV/HIV from the Botswana Combination Prevention Project study (2013-2018) using the Oxford Nanopore GridION platform. Consensus sequences were analyzed for genotypic and mutational profiles. (3) Results: Overall, 98 HBV sequences had evaluable reverse transcriptase region coverage. The median participant age was 43 years (IQR: 37, 49) and 66/98 (67.4%) were female. Most participants, i.e., 86/98 (87.8%) had suppressed HIV viral load (VL). HBV RAMs were identified in 61/98 (62.2%) participants. Most RAMs were in positions 204 (60.3%), 180 (50.5%), and 173 (33.3%), mostly associated with lamivudine resistance. The triple mutations rtM204V/L180M/V173L were the most predominant (17/61 [27.9%]). Most participants (96.7%) with RAMs were on antiretroviral therapy for a median duration of 7.5 years (IQR: 4.8, 10.5). Approximately 27.9% (17/61) of participants with RAMs had undetectable HBV VL, 50.8% (31/61) had VL < 2000 IU/mL, and 13/61 (21.3%) had VL ≥ 2000 IU/mL. (4) Conclusions: The high prevalence of lamivudine RAMs discourages the use of ART regimens with 3TC as the only HBV-active drug in people with HIV/HBV.

Keywords: Africa; Botswana; drug resistance; hepatitis B virus; people living with HIV.