ObjectiveHepatitis B (HB) and C (HC) are two severe viral infectious diseases with a deleterious impact on global health. 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 using immunological and molecular methods.ResultsThe blood samples were obtained from 10,520 enrolled participants. Complete biochemical and hematological tests, as well as urine analysis, were assessed. The presence of HBsAg, anti-HBs, anti-HBc, and anti-HCV antibodies for all participant and HBeAg and anti-HBe antibodies for HB-positive patients were evaluated. Moreover, HB genomic DNA and HC genomic RNA were extracted from serum samples of HB-positive patients. The real-time PCR assay was employed to quantify the gene copies of hepatitis B and C viruses. HC genotyping was also performed. The prevalence of HB and HC was 0.24% (95% CI 0.16–0.35) and 0.11% (95% CI 0.06–0.19), respectively. Rural participants were significantly more HB-positive than the urban people (P = 0.045), while males were significantly more HC-positive than the females (P = 0.013). The prevalence of HB and HC in this area were lower than those of other geographical locations of Iran, which may be due to different lifestyles or other unknown reasons.