Hepatitis B virus (HBV) X protein (HBx) is a viral regulatory and multifunctional protein. It is well-known that the canonical HBx reading frame bears two phylogenetically conserved internal in-frame translational initiation codons at Met2 and Met3, thus possibly generating divergent N-terminal smaller isoforms during translation. Here, we demonstrate that the three distinct HBx isoforms are generated from the ectopically expressed HBV HBx gene, named XF (full-length), XM (medium-length), and XS (short-length); they display different subcellular localizations when expressed individually in cultured hepatoma cells. Particularly, the smallest HBx isoform, XS, displayed a predominantly cytoplasmic localization. To study HBx proteins during viral replication, we performed site-directed mutagenesis to target the individual or combinatorial expression of the HBx isoforms within the HBV viral backbone (full viral genome). Our results indicate that of all HBx isoforms, only the smallest HBx isoform, XS, can restore WT levels of HBV replication, and bind to the viral mini chromosome, thereby establishing an active chromatin state, highlighting its crucial activities during HBV replication. Intriguingly, we found that sequences of HBV HBx genotype H are devoid of the conserved Met3 position, and therefore HBV genotype H infection is naturally silent for the expression of the HBx XS isoform. Finally, we found that the HBx XM (medium-length) isoform shares significant sequence similarity with the N-terminus domain of the COMMD8 protein, a member of the copper metabolism MURR1 domain-containing (COMMD) protein family. This novel finding might facilitate studies on the phylogenetic origin of the HBV X protein. The identification and functional characterization of its isoforms will shift the paradigm by changing the concept of HBx from being a unique, canonical, and multifunctional protein toward the occurrence of different HBx isoforms, carrying out different overlapping functions at different subcellular localizations during HBV genome replication. Significantly, our current work unveils new crucial HBV targets to study for potential antiviral research, and human virus pathogenesis.