Hepatitis B virus (HBV) may integrate into the genome of an infected cell and contribute to hepatocarcinogenesis. In this study, we applied a new high-throughput HBV integration sequencing approach that allows sensitive identification of HBV integration sites and enumeration of integration clones. We identified 3,339 HBV integration sites in paired tumour and non-tumour tissue samples from 7 patients with hepatocellular carcinoma (HCC). A total of 2,107 clonally expanded integrations were detected: 1,817 in tumour and 290 in non-tumour tissues. Moreover, we detected significant enrichment of clonal integrations in mitochondrial DNA (mtDNA), and HBV integration in mtDNA preferentially involved OXPHOS genes in tumours and the regulatory D-loop region in non-tumour tissues. We also found that HBV RNA sequences may be imported into the mitochondria of hepatoma cells, polynucleotide phosphorylase (PNPASE) is involved in this import, and HBV RNA might be involved in the process of HBV integration into mtDNA. Our data suggest a novel mechanism by which HBV insertion might contribute to HCC development.