The prevalence of hepatitis B virus infection among medical professionals is of great concern and this was due to increase in risky activities during clinical training such as needle stick injury, frequent contact with blood and other body fluids; and risky behaviours outside the clinical areas such as having unprotected sex, and injecting drugs. A descriptive cross-sectional study on preclinical students who are yet to be placed or begin practice in the clinical areas was conducted at Kampala International University Teaching Hospital (KIU-TH) in Ishaka sub-county, Bushenyi district in western Uganda between December, 2014 and January, 2015. The purpose of the study was to determine the sero-prevalence of Hepatitis B virus infection among preclinical students of Kampala International University western campus (KIU-WC) and also, to know medical students who were infected before their enrollment in medical school and those who will be found negative; to be vaccinated before going into the clinical areas. A total of one hundred and fifty (150) research participants were recruited for the study using a cluster and simple random sampling technique. Samples were collected from preclinical students of Medicine and surgery, Medical laboratory sciences, Nursing and Pharmacy department. All blood samples collected into a plain tube were allowed to clot and centrifuged to obtain serum; this is because, serum is the most preferred sample in serological testing for antigen and antibody-dependent. Samples were tested for HBsAg with SD BIOLINE United States of America (USA) immune chromatographic rapid test strips with high sensitivity of 99.8% and specificity of 100%. Data on risk factors were collected using a structured closed-ended questionnaire. The prevalence of hepatitis B virus (HBV) infection in this study was 4% which is lower among the preclinical students of Kampala International University (KIU). Fisher exact test was done to determine significance to risk factors in this study were needle stick injury, contact with blood and blood transfusion were significantly associated with HBV infection at p-value of 0.05. There is need for preclinical student to know their HBV status and those tested negative be vaccinated and this will give a clear source of HBV infection among medical students. This is important in order to avoid students’ parents and guidance taking legal action against institutions of learning in the name of infecting their children in the clinical areas during training.