Objective: Hepatitis B (HB) and C (HC) are two severe infectious diseases with a high global health impact. This study aimed to evaluate the prevalence of HB and HC in the Prospective Epidemiological Research Studies of the Iranian Adults (PERSIAN) Guilan Cohort Study through immunological and molecular methods. Results: The blood samples were obtained from 10520 enrolled participants. Complete biochemical and hematological assessments plus urine analysis were done. The presence of HBsAg, anti-HBs, anti-HBc, and anti-HCV antibodies for all participants, and HBeAg and anti-HBe antibodies for HB positive patients were evaluated. HB genomic DNA and HC genomic RNA were extracted from positive serum samples. The real-time PCR assay was done to quantify HB and HC genomes. HC genotyping was also performed. The HB and HC prevalence was 0.24% (95% CI, 0.16 % to 0.35%) and 0.11% (95% CI, 0.06 % to 0.19%), respectively. Rural participants were significantly more HB positive than urban people (P=0.045), while male individuals were significantly more HC positive than female participants (P=0.013). The obtained showed the prevalence of HB and HC in this area were lower than other geographical locations of Iran, which may be due to different lifestyles or other unknown reasons.