Background: Despite international efforts to prevent hepatitis B virus (HBV) infection through global vaccination programs, new cases are still being reported throughout the world. Methods: To supply data that might assist in improving preventive measures and national surveillance for HBV infection, a cross-sectional study was conducted among individuals referred to the Brazilian National Reference Center for Viral Hepatitis (Rio de Janeiro) during a two-year period. Reported risk factors among infected subjects ("HBV-positive") were compared to those of subjects never exposed ("HBV-negative") to HBV. Two subgroups were further identified within the HBV-positive group, "acute" infection and "non-acute" infection. Results: A total of 1,539 subjects were tested for HBV, of which 616 were HBV-positive (79 acute infection and 537 non-acute infection). HBV-positive subjects were more likely to be of male gender (63% versus 47%); and to report multiple sexual partners (12% versus 6%) and illicit drug use (IDU and/or intranasal cocaine use) (6% versus 3%). Among the HBV-positive subgroups, age differed significantly, with 48% being under 30 years of age in subjects acutely infected compared to 17% in those with non-acute infection. Conclusions: The association of multiple sexual partners with past HBV infection and the age distribution of currently infected subjects suggest that sexual transmission played a major role in the transmission of HBV in this study population. Thus, vaccination during adolescence should be considered.