The study was undertaken to investigate the features and clinical implications of hepatitis B virus (HBV) genotypes, basal core promoter (BCP) and precore (PC) mutations in hepatitis B-related acute-on-chronic liver failure (HB-ACLF). Samples from 75 patients with HB-ACLF and without pre-existing liver cirrhosis and 328 age-matched patients with chronic hepatitis B (CHB) were analyzed. HBV genotype and BCP/PC mutations were determined by direct sequencing. Mutations at 8 sites of the BCP/PC region were compared between the two groups of patients. A significantly higher ratio of genotype B to C was found in patients with HB-ACLF than in patients with CHB (30.7–69.3%vs16.5–82.6%, P < 0.01). Single mutations including T1753V (C/A/G), A1762T, G1764A, G1896A and G1899A and triple mutations T1753V/A1762T/G1764A and A1762T/G1764A/C1766T (or T1768A) were more frequently detected in patients with HB-ACLF than in patients with CHB. Correspondingly, BCP/PC wild-type sequences were absent in patients with HB-ACLF in contrast to 27.1% in patients with CHB. The BCP/PC mutations were found to be associated with increased HBeAg negativity, higher alanine aminotransferase level and lower viral load. Patients with HB-ACLF infected with the PC mutant virus had a higher mortality. The findings suggest that patients with CHB infected with genotype B with BCP/PC mutations were more likely to develop HB-ACLF than those with genotype C with wild-type BCP/PC regions, and patients with HB-ACLF with the PC mutation had increased risk of a fatal outcome.