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Developing a vaccine to prevent congenital cytomegalovirus (CMV) infection and newborn disability requires an understanding of infection incidence. In a prospective cohort study of 363 adolescent girls (NCT01691820), CMV serostatus, primary infection, and secondary infection were determined in blood and urine samples collected at enrollment and every 4 months for 3 years. Baseline CMV seroprevalence was 58%. Primary infection occurred in 14.8% of seronegative girls. Among seropositive girls, 5.9% had ≥4-fold increase in anti-CMV antibody, and 23.9% shed CMV DNA in urine. Our findings provide insights on infection epidemiology and highlight the need for more standardized markers of secondary infection., Competing Interests: Potential conflicts of interest. M. D., N. D. S., A. G., A. A. P., F. R., J. Y., and I. D. are employed by and hold shares of GSK. J.-M. D. and L. G. are employees of GSK. R. P. was an employee of GSK at the time of the study; he holds shares of GSK, is currently an employee of Moderna Inc, and holds shares of Moderna Inc. D. A. and S. B. received a grant from GSK for this study. S. B. is a member of advisory committees for Merck, Moderna Therapeutics, and Sanofi, and received grants from Pfizer and Moderna outside the submitted work. E. M. Y. reports no potential conflicts. The authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2023. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)