Hepatitis B virus (HBV) reverse transcriptase (RT) is encoded by the polymerase gene in the reverse transcriptase region, which overlaps with the S gene. The association between mutations of HBV RT and the pathobiological features of hepatocellular carcinoma (HCC) remain to be elucidated. The present study aimed to examine mutations in this region of the HBV genome and its clinical significance. Briefly, HBV total DNA was extracted from 84 pairs of HCC tumor tissue and corresponding adjacent non‑tumor tissue samples. The RT/S regions (nt130‑1161) were amplified and sequenced using the Sanger method, and associations between RT mutations and the clinical characteristics of patients with HCC were analyzed. Finally, 27 and 29 mutations with frequencies >5% were identified in the RT and S regions, respectively. The rtF221Y variation and a tumor size >8 cm were found to be independent risk factors for the postoperative recurrence of HCC, with hazard ratios of 2.345 (95% CI, 1.391‑3.953; P=0.001) and 1.838 (95% CI, 1.069‑3.161; P=0.028), respectively. rtF221Y was also an independent risk factor for poor overall survival rates (HR=2.557; 95% CI, 1.344‑4.866; P=0.004). The mutation of R122 K in the HBV S protein was closely associated with tumor recurrence (P<0.001). As a result, rtF221Y was identified as a risk factor for poor prognosis and may be a potential viral marker for predicting prognosis in HCC.