During chronic hepatitis B virus (HBV) infection, double substitution mutations in the basal core promoter (BCP) region frequently emerge that include A1762T/G1764A and the neighbouring C1766T/T1768A mutations, here termed BCP1 and BCP2, respectively. Due to a compact viral genome organization, BCP1 and BCP2 mutations result in amino acids changes in the overlapping X gene: K130M/V131I and F132Y, respectively. It has been shown that both BCP mutations lead to a modest increase in viral genome replication. However, the question of whether the alteration that occurs in the overlapping X gene might contribute to the increased viral genome replication has not been properly addressed. This study genetically separated the core promoter from the overlapping X gene using 1.3mer overlength HBV constructs and examined the impact of the X gene mutations on viral genome replication in HepG2 cells. Each BCP mutation resulted in modestly enhanced viral genome replication that occurred via augmented viral transcription. Therefore, it was concluded that these BCP mutations do not affect expression of the overlapping X gene or impair its stimulatory effect on viral genome replication.