Hepatocellular carcinoma (HCC) is a major cause of cancer morbidity and mortality in many parts of the world, including Asia and sub-Saharan Africa, where there are upwards of 600,000 new cases each year. Over 80% of the burden of HCC is manifest in the economically developing world. Further, the median age of diagnosis and death from HCC is between 45 and 55 years of age in these regions. Since the occurrence of HCC is coincident with regions where aflatoxin exposure is high, efforts started in the 1960s to investigate this possible association. Aflatoxin biomarkers of internal and biologically effective dose have been integral to definitively establish the etiologic role of this toxin in human HCC. In two separate cohort studies, a strong multiplicative relationship between aflatoxin exposure and the hepatitis B virus for the development of HCC was found. Further, in recent years, research has demonstrated that aflatoxin exposure is also linked to the occurrence of a specific mutation in the p53 tumor suppressor gene. Thus, the development and application of molecular biomarkers reflecting events from exposure to manifestation of clinical disease has rapidly expanded our knowledge of the mechanisms of disease pathogenesis in HCC, and this will have increasing potential for early detection, treatment, interventions, and prevention.