The protein encoded by the hepatitis B virus (HBV)-X gene, HBX, has been implicated to be involved in the development of HBV-associated liver cancer. HBX is a multifunctional regulatory protein that has been identified as a potential oncogene but its exact function remains unclear. HBX was documented to interact with several factors involved in cellular DNA repair as well as compromise the cell's ability to repair damaged DNA. We previously documented an accumulation of genetic alterations in two HepG2 cell lines independently transfected with HBV. In this report, we investigate the effect of the HBV-X gene (HBX) on the stability of the host genome using HepG2 stable transfectants (HepG2-HBX) and vector controls (HepG2-neo). We document that all HepG2-HBX clones analyzed contain HBX gene integrated and HBX transcript. Our data demonstrate that HepG2-HBX cells have an increased number of chromosome alterations and micronuclei formation compared to vector controls. A total of 10 de novo chromosomal rearrangements involving nine different chromosomes were detected in the HepG2-HBX clones, while no new rearrangements were found in vector controls. Each HepG2-HBX clone contained independently occurring de novo alterations not found in other HBX or vector clones. A three-fold increase of micronuclei formation was detected in HepG2-HBX cells compared to vector controls. Micronuclei originated from all chromosomes, however, preliminary data indicated that micronuclei originating from chromosomes 2, 3, 7, 18 and 20 were found in a greater amount in cells expressing the HBX gene. Interestingly, chromosomes 2, 18 and 20 were three of the chromosomes found rearranged in HepG2-HBX clones. These data provide evidence that genomic integrity was affected in cells expressing the HBX gene. De novo cytogenetic alterations identified in HepG2-HBX clones implicate the involvement of HBX in the process and support the hypothesis that HBX may interfere with normal cellular processes responsible for genomic integrity, increasing the risk for acquiring genetic mutations in infected hepatocytes.