Abstract: (1) Background: We determined the prevalence of hepatitis B virus (HBV) amongst people without human immunodeficiency virus (HIV) in rural and peri-urban areas in Botswana. (2) Methods: We screened hepatitis B surface antigen (HBsAg) from archived plasma samples of people without HIV (n=2135) randomly selected from the Botswana Combination Prevention Program (BCPP) (2013–2018). We sequenced 415 bp of the surface region using BigDye sequencing chemistry. (3) Results: The median age of participants was 31 (IQR: 24–46) and 64% (1360/2135) were female. HBsAg prevalence was 4.0% (86/2135) [95% CI: 3.3 – 4.9]) and ranged between 0%–9.2%. Older participants (>35 years) had increased odds of HBsAg positivity (OR: 1.94; 95% CI: [1.32–2.86]; p = 0.001. Thirteen samples were sequenced and 7 (53.8%) were genotype A, 3 (23.1%) were genotype D and genotype E each. Clinically significant mutations were identified in the surface region, but no classic drug resistance mutations were identified. (4) Conclusions: We report an HBV prevalence of 4.0% (95% CI 3.3 – 4.9) among people without HIV in rural and pe-ri-urban communities in Botswana with varying rates in different communities. A comprehen-sive national HBV program is required in Botswana to guide HBV prevention, testing and management.