Abstract 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.