This study investigated hepatitis B virus (HBV) single nucleotide variants (SNVs) and deletion mutations linked with hepatocellular carcinoma (HCC). Ninety-three HCC patients and 108 non-HCC patients were enrolled for HBV genome-wide next-generation sequencing (NGS) analysis. Systemic literature review and meta-analysis were performed to validate NGS-defined HCC-associated SNVs and deletions. The experimental results identified 60 NGS-defined HCC-associated SNVs, including 41 novel SNVs, and their pathogenic frequencies. Each SNV was specific for either genotype B (n=24) or genotype C (n=34), except nt53C for both genotypes. The pathogenic frequencies of these HCC-associated SNVs exhibited a distinct U-shaped distribution pattern. According to meta-analysis and literature review, 167 HBV variants from 109 publications were categorized into 4 levels (A–D) of supporting evidence associated with HCC. The proportion of NGS-defined HCC-associated SNVs among these HBV variants declined significantly from 75% of 12 HCC-associated variants by meta-analysis (Level A) to 0% of 10 HCC-unassociated variants by meta-analysis (Level D) (P<0.0001). PreS deletions were significantly associated with HCC, in terms of deletion index, for both genotypes B (P=0.030) and C (P=0.049). For genotype C, preS deletions involving a specific fragment (nt2977–3013) were significantly associated with HCC (HCC vs non-HCC, 6/34 vs 0/32, P=0.025). Meta-analysis of preS deletions showed significant association with HCC (summary odds ratio: 3.0, 95% confidence interval, 2.3 to 3.9). Through transfecting Huh7 cells, all the five novel NGS-defined HCC-associated SNVs in the small surface region influenced hepatocarcinogenesis pathways, including ER-stress and DNA repair systems, by microarray, real-time PCR and western blot analysis. Their carcinogenic mechanisms are worthy of further research.