In Colombian blood banks, screening for the surface antigen of hepatitis B is mandatory in all units collected. Testing of antibody against core antigens is not administered, although this method may be useful to detect donors infected with the hepatitis B virus.The prevalence of occult hepatitis B was determined by applying a full-serological profile of hepatitis B virus to blood samples of blood donors.Between April 2008 and October 2009, a prospective cross sectional study was conducted using 628 samples from donors to blood banks located in four Colombian cities. Prevalence for hepatitis B had been previously recorded for these cities. Serological screening was performed for the complete virus; then nucleic acid amplification was tested in sera that were anti-HBc reactive and with a titer of anti-HBs ≤30 mUI/ml.Of the 628 samples tested, 129 met the serological criteria established to be tested nucleic acid amplification. None of them demonstrated evidence of nucleic acid amplification of hepatitis B virus.This is the first study in Colombia to detect the presence of blood donors that may be occult hepatitis B carriers. None was detected.