Mutations within the hepatitis B virus surface antigen (HBsAg) were found to correlate with progressive liver diseases, including hepatocellular carcinoma (HCC). Mutations in this region can impact viral morphogenesis, virus-host interactions, and immune responses. In this cross-sectional study, we screened for mutations in the pre-S/S regions of HBsAg in sequences retrospectively generated from samples collected in Saudi Arabia. We analyzed 304 full-length HBsAg sequences isolated from samples collected from four clinical groups: inactive (n = 180), active (n = 62), liver cirrhosis (LC) (n = 36), and HCC (n = 26). Three mutations (N103D, Q30K, and I208T) in HBsAg showed significantly higher frequencies in the HCC group compared to other clinical groups. Additionally, the presence of the three mutations combined was significantly associated with HCC in a multivariate analysis. The evolutionary analysis further revealed that these mutation sites are subjected to positive selection within the HCC group. The structural analysis suggested that position 103 within HBsAg pre-S region is prominently accessible and mutations at this site may disrupt interactions with viral/cellular factors or impact recognition by immune responses. Collectively, our findings highlight a significant increase in the frequency of three HBsAg mutations in a cohort of HCC patients in Saudi Arabia and their potential effect.