Mutations in the basal core promoter (BCP) and precore (PC) regions of the hepatitis B virus (HBV) are more common in genotypes B and C than in genotype A, suggesting that these mutations might affect replication competency depending on genotype. The purpose of the study was to investigate the influence of these mutations on the capacity of HBV for replication and antiviral drug susceptibility according to genotype. Genotypes A, B, and C of HBV strains with a BCP mutation, PC mutation, or BCP + PC mutation were made by site-directed mutagenesis. Replication competency of each construct and susceptibility to nucleos(t) ide analogues were tested in an Huh7 cell line. In genotype A, the BCP and BCP + PC mutations increased the viral replication around 6.5 times compared with the wild type, and the PC mutation alone similarly increased the viral replication around three times. In genotypes B and C, all three mutant types increased viral replication to a similar extent, regardless of mutation pattern. Interestingly, the BCP mutation appeared to have a greater effect on viral replication in genotype A than in genotypes B and C. This finding was unexpected because the BCP mutation is more common in HBV genotypes B and C. Moreover, the BCP, PC, and BCP + PC mutations decreased the sensitivity of HBV to antiviral agents to various degrees (2- to 10-fold) regardless of genotype. In conclusion, BCP and PC mutations increased viral replication regardless of HBV genotype and decreased in vitro antiviral susceptibility to the nucleos(t) ide analogues. J. Med. Virol. 87:601–608, 2015. © 2015 Wiley Periodicals, Inc.