There is significant regional variation in the etiologic agents responsible for hepatocellular carcinoma (HCC), which influences the genetic and epigenetic mechanisms of malignant transformation. The aim of the present study was to investigate the prevalence of allelic imbalance (AI) and CpG island methylation in HCCs from Australia and South Africa. Genomic DNA was extracted from malignant and non-malignant liver from 37 Australian and 24 South African HCCs and histologically normal liver from 20 transplant donors. AI was examined at 1p, 4p, 4q, 8p, 9p, 13q, 16q and 17p, using 23 microsatellite markers. Methylation status of p14, p16, p15, RIZ1, E-cadherin and O6-MGMT was examined using methylation specific PCR, while MINTs 1, 2, 12, 25 and 31 were assessed using combined bisulfite restriction analysis. The highest prevalence of AI was observed at 9p (69%) and 17p (52%). AI was significantly higher in South African HCCs (p<0.05). The prevalence of promoter methylation of the six genes was significantly higher in Australian cases in both malignant and non-malignant liver tissue (p<0.05). MINT assays revealed an increasing degree of CpG island methylation in the progression of hepatocarcinogenesis which was significant for MINTs 1, 12 and 31 (p<0.05). MINT methylation was more prominent in Australian HCCs. These data indicate that methylation is an early event preceding malignant transformation. Methylation was more and AI less prevalent in Australian than South African HCCs. These data suggest that there are different mechanisms of malignant transformation in HCCs from Australia and South Africa.