Background: Hepatitis B virus (HBV) sequencing data are important for monitoring HBV evolution. We aimed to molecularly characterize HBV sequences from participants with HBV surface antigen-positive (HBsAg+) serology and occult hepatitis B infection (OBI+). (2) Methods: We utilized archived plasma samples from people living with human immunodeficiency virus (PLWH) in Botswana. HBV DNA was sequenced, genotyped and analyzed for mutations. We compared mutations from study sequences to those from previously generated HBV sequences in Botswana. The impact of OBI-associated mutations on protein function was assessed using the Protein Variation Effect Analyzer. (3) Results: Sequencing success was higher in HBsAg+ than in OBI+ samples [86/128 (67.2%) vs. 21/71 (29.2%)]. Overall, 93.5% (100/107) of sequences were genotype A1, 2.8% (3/107) were D3 and 3.7% (4/107) were E. We identified 13 escape mutations in 18/90 (20%) sequences with HBsAg coverage, with K122R having the highest frequency. The mutational profile of current sequences differed from previous Botswana HBV sequences, suggesting possible mutational changes over time. Mutations deemed to have an impact on protein function were tpQ6H, surfaceV194A and preCW28L. (4) Conclusions: We characterized HBV sequences from PLWH in Botswana. Escape mutations were prevalent and were not associated with OBI. Longitudinal HBV studies are needed to investigate HBV natural evolution.

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