INTRODUCTION AND OBJECTIVES: Chronic hepatitis B virus (HBV) infection is a major risk factor for hepatocellular carcinoma (HCC). In African populations, HCC frequently presents at an advanced stage with poor outcomes. We applied whole genome sequencing (WGS) to compare HBV genomes in individuals with and without HCC. MATERIALS AND METHODS: We identified adults with HBV infection, with and without complicating HCC, in Cape Town, South Africa. We generated HBV WGS using pan-genotypic probe-based enrichment followed by Illumina sequencing. RESULTS: Compared to the non-HCC group, HCC patients were more likely to be male (p<
0.0001), older (p=0.01), HIV-negative (p=0.006), and have higher HBV viral loads (p<
0.0001). Among 19 HCC and 12 non-HCC patients for whom WGS was obtained, genotype A dominated (74%), of which 96% were subgenotype A1. PreS2 deletions (Δ38-55) were enriched in HBV sequences from HCC patients (n=7). The sequence motif most strongly associated with HCC comprised either a deletion or polymorphism at site T53 in PreS2 - collectively coined 'non-T53' - together with a basal core promoter (BCP) mutation G1764A (AUROC = 0.79). CONCLUSIONS: In this setting, HBV sequence polymorphisms and deletions are associated with HCC, and 'non-T53+G1764A' represents a putative signature motif for HCC. Additional investigations are needed to disaggregate the impact of other demographic, clinical, and environmental influences, to ascertain the extent to which viral polymorphisms contribute to oncogenesis, and to determine whether HBV sequence is a useful biomarker for HCC risk stratification.