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Background: Hepatitis B infection is the major risk factor for liver cancer in Iran. There is no comprehensive population-based study on the prevalence of hepatitis B by regional distribution. Moreover, systematic reviews of hepatitis B prevalence lack knowledge of some regions. We aimed to estimate the prevalence of hepatitis B and its temporal trends over 17 years by sex, age and geographical distribution. Methods: We used the Iranian Blood Donors data in addition to systematic reviews on population-based studies at national and provincial levels and statistical methods (A two-stage spatio-temporal model and crosswalk approach) to address the missing points of hepatitis B prevalence among the general population. The direct age-standardized approach was applied using Iran's national population in 2016. Results: At national level, age-standardized hepatitis B prevalence in Iran decreased from 3.02% (95% uncertainty interval; 2.26 to 3.96) in 2000 to 1.09% (95% uncertainty interval; 0.85 to 1.37) in 2016, with a total -64.84% change. Hepatitis B prevalence was more than 1.3 times greater in males than females in 2016. Overall, the prevalence of hepatitis B increased with increasing age. At provincial level, in 2016, the province with the highest prevalence had a nearly 11-time greater rate compared to the lowest prevalence. The declining annual percent change (APC) of the prevalence trend varied between -11.53% to -0.5% at provincial level from 2000 to 2016. Only one province did not witness a downward trend in which the APC was 0.5% (95% UI:0.47-0.54). Conclusion: The downward trend in prevalence of hepatitis B infection indicates the effectiveness of strategies and preventive measures adapted in Iran. Nevertheless, we need to eradicate this infection. In this regard, re-evaluating preventive measures, especially in high-risk age groups of the population, is recommended. [ABSTRACT FROM AUTHOR]