This study explored the combined effect of number and pattern of mutations in the X/precore regions of the hepatitis B virus (HBV) genome, mutational complex genotype (MCG), on hepatocellular carcinoma (HCC) development. Sequence variations were determined by direct sequencing and multiplex restriction fragment mass polymorphism analysis in 150 age-, sex- and hepatitis B e antigen (HBeAg) status-matched patients with and without HCC. In addition, a longitudinal study and an external validation of MCG were conducted. All were HBV subgenotype C2. Eight high-frequency mutations (G1613A, C1653T, T1753V, A1762T, G1764A, A1846T, G1896A and G1899A) were significantly associated with HCC. Whereas C1653T, T1753V, G1764A and A1846T were independent mutational factors for HCC, the significance of these individual mutations was negligible when analyzed with all clinico-virological variables. The total number of mutations was the only independent viral factor for HCC, irrespective of HBeAg status. There was a significant dose–risk relationship between the number of mutations and HCC, in which high risks for HCC were associated with mutation numbers ≥6. Pattern analysis of the mutations revealed disparity in distribution among the top seven high-risk mutation combination patterns, which accounted for 40 and 2.7% of HCC and non-HCC cases, respectively. The predictive accuracy of the high-risk mutations for HCC was similar to that of α-fetoprotein. Longitudinal and external validation studies also supported the association of mutation number with HCC development. MCG in the HBV X/precore regions is a risk indicator for HCC, and might serve as a new guide to the HCC screening scheme for chronic HBV carriers. (Cancer Sci 2012; 103: 296–304)