Hepatitis B virus (HBV) infection is a potentially life-threatening infection that attacks the liver and can cause both acute and chronic disease. This creates a high risk of death from cirrhosis and liver cancer. Hepatitis B infection poses a major health concern globally. It is estimated that 257 million people are infected globally with 780,000 deaths reported annually. In Kenya, HBV prevalence stands at chronic states of intermediate range (5% -7%) and high (≥8%) with regional variations. Garissa County carries a high HBV infection risk with a reported prevalence of 14.1% in pregnant women attending antenatal care (ANC) clinics. This study was carried out to determine and compare the seroprevalence of HBV among in-mates and voluntary blood donors at Garissa Main Prison and Garissa County referral hospital respectively in Garissa, Kenya. A total of 130 in-mates and 130 voluntary blood donors were sampled in this study. Serum was tested for Hepatitis B surface antigen (HBsAg) using a rapid test cassette (Amitech Diagnostics Inc.). A questionnaire was also used to collect socio-demographic factors of the study participants. Data were entered and analyzed using SPSS version 20. Majority of the study participants were males (86.9% among inmates and 95.4% among blood donors). Majority (76.2%) of the in-mates and of the donors (83.1%) were aged between 20 -40 years while majority (51.4% of the donors and 81.5% of in mates) had only a primary school level of education. HBV seroprevalence was significantly higher among in mates compared to blood donors. Out of the total number of in-mates tested, 7 (5.4%) were HBV seropositive. Conversely, among blood donors 4 (3.1%) were seropositive. There was a significant association between HBV seropositivity and gender among both the blood donors and in-mates. There was no significant association between HBV seropositivity and both level of education and age. No data currently