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Background: The first large serosurvey in Iran found a SARS-CoV-2 antibody seroprevalence of 17.1% among the general population in the first wave of the epidemic by April, 2020. The purpose of the current study was to assess the seroprevalence of COVID-19 infection among Iranian general population after the third wave of the disease., Methods: This population-based cross-sectional study was conducted on 7411 individuals aged ≥10 years old in 16 cities across 15 provinces in Iran between January and March, 2021. We randomly sampled individuals registered in the Iranian electronic health record system based on their national identification numbers and invited them by telephone to a healthcare center for data collection. Presence of SARS-CoV-2-specific IgG and IgM antibodies was assessed using the SARS-CoV-2 ELISA kits. The participants were also asked about their recent COVID-19-related symptoms, including cough, fever, chills, sore throat, headache, dyspnea, diarrhea, anosmia, conjunctivitis, weakness, myalgia, arthralgia, altered level of consciousness, and chest pain. The seroprevalence was estimated after adjustment for population weighting and test performance., Results: The overall population-weighted seroprevalence adjusted for test performance was 34.2% (95% CI 31.0-37.3), with an estimated 7,667,874 (95% CI 6,950,412-8,362,915) infected individuals from the 16 cities. The seroprevalence varied between the cities, from the highest estimate in Tabriz (39.2% [95% CI 33.0-45.5]) to the lowest estimate in Kerman (16.0% [95% CI 10.7-21.4]). In the 16 cities studied, 50.9% of the seropositive individuals did not report a history of symptoms suggestive of COVID-19, implying an estimation of 3,902,948 (95% CI 3,537,760-4,256,724) asymptomatic infected individuals., Conclusions: Nearly one in three individuals were exposed to SARS-CoV-2 in the studied cities by March 2021. The seroprevalence increased about two-fold between April, 2020, and March, 2021., (© 2022. The Author(s).)