Background and aim In China, acute-on-chronic liver failure (ACLF) is mostly caused by hepatitis B virus (HBV). However, the mechanism remains unclear. This study aims to investigate the association between both HBV genotype and mutations in basal core promoter (BCP) and pre-core/core (pre-C/C) regions with the development of HB-ACLF. Methods A multicenter cross-sectional study was performed in China. Serum samples from 522 patients were analyzed, including 231 patients with mild-chronic hepatitis B (CHB-M), 84 with severe-chronic hepatitis B (CHB-S) and 207 with HB-ACLF. HBV genotype and related mutations in the BCP and pre-C/C regions were determined by direct sequencing. Results A significantly higher ratio of HBV genotype B to C was detected in HB-ACLF patients than in CHB-M or CHB-S patients. The A1762T/G1764A, A1846T and G1896A mutations were significantly more common in HB-ACLF patients infected with either genotype B or C as compared with CHB-M, whereas the C1913A/G and A2159G mutations were more associated with HB-ACLF in genotype C patients. Comparing with CHB-S, the A1762T/G1764A mutation in genotype B and the A2159G mutation in genotype C were significantly more common in HB-ACLF patients. A multivariate analysis showed that factors such as HBV genotype B, age >= 40 years and A1762T/G1764A, A1846T and G1896A mutations were independently associated with the development of HBA-CLF. Conclusion Chronic HBV infection with genotype B, A1762T/G1764A, A1846T and G1896A mutations has a higher possibility to develop HB-ACLF. These virological factors could serve as possible molecular markers for prediction of the clinical outcomes of chronic HBV infection.