Primary hepatocellular carcinoma (HCC) is one of the most common cancers worldwide. Hepatitis B virus (HBV) and aflatoxin exposure represent the main risk factors for the development of HCC in areas endemic for liver cancer. The recently developed individual biological markers of aflatoxin exposure permit a better quantitative estimation of aflatoxin exposure in different populations of the world. Research into the association between aflatoxin and HCC represents one of the most complete examples of the use of biomarkers to assist in the identifications of risk factors associated with carcinogensis in humans. This paper reviews the data that have accumulated from both traditional and molecular epidemiological studies implicating aflatoxin exposure as an important risk factor in the etiology of HCC. In addition, epidemiological and experimental data are examined regarding the synergistic effects of aflatoxin exposure and HBV on HCC formation and the predominance of one hotspot GC→TA transversion in the p53 gene of aflatoxin-induced hepatocarcinogenesis.