Background: Occult hepatitis B virus infection (OBI) is characterized by the presence of HBV DNA in individuals who test negative for hepatitis B surface antigen (HBsAg). It poses diagnostic challenges and contributes to chronic liver diseases. Understanding its epidemiology, pathogenesis and clinical outcomes remains essential. This study aimed to assess the prevalence and characteristics of OBI in Northwestern Greece. Methods:Serum samples were prospectively collected from 702 blood donors at the University Hospital of Ioannina, Greece, between February 2018 and September 2022. The investigation focused on the presence of hepatitis B virus (HBV) markers, utilizing the Abbott Architect HBsAg and HBcAb HB Qualitative II kit for the detection of HBsAg and anti-HBc, respectively. Further analysis was conducted on serum samples from individuals who tested negative for HBsAg but positive for anti-HBc, employing polymerase chain reaction (PCR) to detect HBV-DNA. In instances of OBI, sequencing and mutation analysis of the HBV pre-S/S gene were carried out for comprehensive characterization. Results:Screening revealed 56 cases (7.9%) with active HBV infection (HBsAg positive) and identified 144 cases (20.5%) indicative of past HBV infection (HBsAg negative, anti-HBc positive). Additionally, a prevalence of 5.4% (38/702) of OBI was detected. Among these, 36 cases exhibited a low HBV DNA load of less than 225 IU/mL. Notably, one OBI patient was co-infected with HIV. Furthermore, two cases of OBI with high HBV-DNA levels exceeding 200,000 IU/ml were detected. Sequencing analysis unveiled S- and pre-S mutations in four cases of OBI, including both instances with elevated HBV-DNA levels. Conclusion:In a region with a high proportion of immigrants from countries where HBV is endemic, a high prevalence of HBV infection and occult HBV infection (OBI) has been detected. Furthermore, mutations in the S gene were found to be associated with cases of OBI with high levels of HBV-DNA. However, additional research is needed to validate the results and understand the clinical relevance of specific OBI mutations for disease progression and treatment efficacy.