Abstract Background Infectious mononucleosis (IM) is a contagious illness most frequently caused by Epstein-Barr virus (EBV). There are no recent published studies characterizing the burden of IM in the general US population over time. Methods Nationally representative samples of US ambulatory medical care data were utilized to estimate the N and proportion of visits with an IM diagnosis (based on ICD-9/10 codes) by time period. The National Ambulatory Medical Care Survey (NAMCS: 2006–2015) and National Hospital Ambulatory Care Survey (NHAMCS: 2006–2019) allow estimation of annual visits in free-standing and hospital-based ambulatory care, including emergency departments (EDs). Restricted cubic spline models were implemented to assess temporal trends. NHAMCS data were divided based on availability: combined outpatient and ED (2006–2011) and ED-only (2012–2019). Analyses were conducted using SAS 9.4 & SUDAAN 11.0.3. Results There were 3,182,082 IM-related visits in NAMCS, 599,837 in combined outpatient and ED NHAMCS, and 449,000 in ED-only NHAMCS across study years. For overlapping study years, 2006–2011, across all databases the average annual number of IM-related visits was 419,927. Persons aged 10–30 years comprised the majority of IM-related visits: 61% in NAMCS, 88% in combined NHAMCS, and 85% in ED-only NHAMCS. While 66% of IM visits were made by females in NAMCS, this proportion was lower in combined NHAMCS (58%) and ED-only NHAMCS (51%). Most IM-related visits were among non-Hispanic white patients: 92% in NAMCS, 87% in combined NHAMCS, and 67% in ED-only NHAMCS. Across databases and years, there were no consistent linear trends in IM-related visits (Figure 1). Rates of IM-related visits in NAMCS decreased from 4.18 (95% confidence interval [CI]: 3.02–5.78) in 2006–2009 to 2.89 (95% CI: 1.40–5.96) per 10,000 visits in 2013–2015; decreased from 4.94 (95% CI: 3.41, 7.16) in 2006–2007 to 3.56 (95% CI: 2.33–5.45) per 10,000 visits in 2010–2011 in combined NHAMCS data; and increased from 3.61 (95% CI: 2.30–5.67) in 2012–2014 to 5.98 (95% CI: 3.20–11.17) per 10,000 visits in 2018–2019 in ED-only data. Conclusion This study contributes epidemiologic information concerning IM and EBV by estimating US ambulatory medical care burden of IM over time and describing the demographics of those affected. Disclosures Christina Chandra, MPH, Moderna, Inc.: Contractor Jianheng Li, MBBS, MPH, Moderna, Inc.: Contractor W. Dana Flanders, MD, DSc, MPH, MA, Moderna, Inc.: Contractor Roderick McPhee, MD, PhD, Moderna, Inc.: Salary|Moderna, Inc.: Stocks/Bonds Robert Paris, MD, Moderna, Inc.: Salary|Moderna, Inc.: Stocks/Bonds Philip Buck, PhD, MPH, Moderna, Inc.: Salary|Moderna, Inc.: Stocks/Bonds Sumana Chandramouli, PhD, Moderna, Inc.: Salary|Moderna, Inc.: Stocks/Bonds Susan Hall, PhD, MS, Moderna, Inc.: Salary|Moderna, Inc.: Stocks/Bonds Cathy Lally, MSPH, Moderna, Inc.: Contractor.