Abstract: Background and Aim The hepatitis B virus (HBV) prevalence study performed in 2003 in Belgium is believed to be underestimating HBV prevalence due to underrepresentation of the non-Belgian population. Therefore, we assessed the prevalence and risk factors of HBV infection in a multi-ethnic region situated in Middle-Limburg Belgium, in 2017. Methods Between May and November 2017, blood samples and questionnaires were taken from patients who presented at the emergency department of a large educational hospital. Blood samples were tested for hepatitis B surface antigen (HBsAg) and hepatitis B core antibodies (anti-HBc). A sample size of 1000 persons was required to obtain a representative sample of the general Middle-Limburg population. Results Of the 1131 patients screened, the overall HBsAg prevalence was 0.97% with differences between Belgians (0.67%) and first-generation-migrants (2.55%), (P = 0.015). Five (45.5%) of 11 HBsAg-positive individuals were not aware of their HBV status. All five (100%) newly diagnosed HBsAg-positive patients had further clinical evaluation and all had a normal level of alanine aminotransferase (ALT). The prevalence of anti-HBc was 8.4%, and was significantly associated with age-gender-ethnicity interaction, presence of HBV-infected household member, hepatitis C virus infection, men who have sex with men, and hemodialysis. Conclusions In this area with large immigrant populations, we found a higher prevalence of HBV infection compared with the nationwide study of 2003. National HBV screening for first-generation migrants is needed as this high-risk group will go unnoticed due to the possible incorrect interpretation of normal ALT values.