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Prevention of Chlamydia trachomatis infection is an ideal application for a vaccine program, which should optimally be administered before sexual debut. However, there are limited epidemiologic studies of C. trachomatis infection in an unselected pediatric population since routine screening and treatment of pregnant women was implemented in the United States in 1993.Anonymized serum samples were obtained from children younger than 21 years in 2 medical centers in Brooklyn, New York, from 2013 to 2015. Anti–C. trachomatis IgG antibody was determined by a validated enzyme immunoassay. Infants younger than 1 year were excluded from the final analysis due to interference of maternal antibody.One thousand two sera were included in the final analysis. Fifty-seven percent were females. No antibody was detected at younger than 11 years. Anti–C. trachomatis IgG antibody was detected in 11.4% and 5.6% of female and male subjects, respectively, older than 11 years (P = 0.0027), and seropositivity increased with age. There was no significant difference in the distribution of age at infection between the centers (P = 0.432), but a difference was detected between genders (P = 0.012) with a higher percentage of female subjects testing positive.Antibody was first detected at 11 years of age, likely coinciding with sexual debut. The prevalence of antibody was higher and appeared earlier in females, mirroring national surveillance trends based on nucleic acid amplification testing. The delay in male antibody detection may be due to biological or behavioral differences between genders. These data are critical in informing potential C. trachomatis vaccine strategies.