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Two plasmid gene protein (Pgp3)-based serological assays, the Pgp3-ELISA and multiplex bead assay (Pgp3-MBA), were compared and used to estimate seropositivity of Chlamydia trachomatis (CT) among females 14 to 39 years old participating in the National Health and Nutrition Examination Survey between 2013–2016. Of the 2,201 specimens tested, 502 (29.5%, 95% CI 27.6–31.5) were positive using Pgp3-ELISA and 624 (28.4%, 95% CI 26.5–30.3) were positive using Pgp3-MBA. The overall agreement between the assays was 87.7%. Corresponding nucleic acid amplification test (NAAT) results were available for 1,725 specimens (from women 18–39 years old); of these, 42 (2.4%, 95% CI 1.8–3.3) were CT NAAT-positive. Most of the CT NAAT-positive specimens had corresponding positive serological assay results; 33 (78.6%, 95% CI 62.8–89.2) were Pgp3-ELISA-positive and 36 (85.7%, 95% CI 70.8–94 …