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Background In contrast to studies that relied on volunteers or convenience sampling, there are few population-based SARS-CoV-2 seroprevalence investigations and most were conducted early in the pandemic. The health department of the fourth largest city in the U.S. recognized that sound estimates of viral impact were needed to inform decision-making. Methods Adapting standardized disaster research methodology in September 2020, the city was divided into high and low strata based on RT-PCR positivity rates, and census block groups within each stratum were randomly selected with probability proportional to size, followed by random selection of households within each group. Using two immunoassays, the proportion of infected individuals was estimated for the city, as well as by positivity rate and by sociodemographic and other characteristics. The degree of under ascertainment of seroprevalence was estimated based on RT-PCR positive cases. Results Seroprevalence was estimated to be 14% with a near two-fold difference in areas with high (18%) versus low (10%) RT-PCR positivity rates and was four times higher compared to case-based surveillance data. Conclusions Seroprevalence was higher than previously reported and is greater than that estimated from RT-PCR data. Results will be used to inform public health decisions about testing, outreach, and vaccine rollout.