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Objectives: Chronic hepatitis B virus (HBV) infection is a lifelong infection that can cause serious liver damage and liver cancer. The last surveillance-based prevalence estimate of chronic HBV infection in New York City was 1.2% in 2008; however, it did not account for persons with undiagnosed infection. The objective of this study was to calculate the prevalence of chronic HBV infection, including undiagnosed infection, for 2016 by using surveillance data and literature-based information., Methods: We calculated the number of persons with diagnosed chronic HBV infection (2000-2016) who were alive and living in New York City in 2016 by using routine surveillance data. We estimated the percentage of persons with undiagnosed chronic HBV infection by using birth region-specific percentages from the literature, weighted by the proportion of the New York City population with diagnosed chronic HBV infection from the same birth region. We identified minimum, maximum, and most likely values for the percentage with undiagnosed chronic HBV infection to generate 95% certainty limits (CLs) of the prevalence estimate., Results: The prevalence of chronic HBV infection in 2016, including undiagnosed infection, in New York City was 2.7% (95% CL, 2.2%-3.6%), representing approximately 230 000 persons. The prevalence of diagnosed chronic HBV infection was 1.5%. The estimated prevalence among non-US-born residents was 6.9% (95% CL, 5.4%-8.9%)., Conclusions: The current burden of chronic HBV infection in New York City, especially for non-US-born residents, is substantial. A renewed focus and dedication of resources is required to increase the number of new diagnoses and improve provider capacity to care for the large number of persons with chronic HBV infection.