Objective To identify the difference and significance of dominant types of hepatitis B virus (HBV) pre-S mutation between liver tumor tissues and paired adjacent non-tumor tissues and to test if the mutations were tumor tissue specific. Methods HBV DNA isolated from 34 paired intratumoral and peritumoral tissues of hepatocellular carcinoma (HCC) patients were screened by PCR and direct sequencing. All patients carried HBV with genotype C, except for one B/C heterozygote. The expression, localization and excretion of LHBs mutant carrying pre-S deletions were characterized in vitro. The expression of endoplasmic reticulum (ER) GRP78 mRNA was assayed. Results Four patterns of pre-S mutations were identified: pre-S1 in-frame deletion involving the first start codon; pre-S2 in-frame deletion; pre-S2 start codon mutation with or without in-frame deletion; and S promoter in-frame deletion (ΔSP). The first two types were evenly found in both tumor and non-tumor tissues. They were rarely present as dominant strains. The last two types were frequently found in the dominant strains in tumor tissues. The overall prevalence of HBV carrying ΔSP was 17.64% (6/34) in tumor tissues, but none were dominant in nontumor tissues. HBV carrying ΔSP was unable to produce S protein in vitro. Immunocytofluorescence assay showed that the ΔSP LHBs mutant aggregated in the cytoplasm, accumulating mainly in the ER. Transient transfection and expression of ΔSP mutant caused GRP78 up-regulation in vitro. Conclusions HBV S promoter deletion was found dominantly in HCC tumor tissue. The aggregation of mutant large surface proteins in the ER possibly involved in HBV-related HCC.