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Abstract Serological surveillance for trachoma could allow monitoring of transmission levels in areas that have achieved elimination targets. Platforms that allow testing in basic laboratories or testing of easy-to-manage samples such as dried blood spots would contribute to the feasibility of serologic testing. Blood from 506 1–12-year-olds in 2 villages in Kongwa district, Tanzania, was tested for antibodies against the antigen Pgp3. Whole blood, plasma, and dried blood spots (DBS) were tested in lab and field settings using a cassette-enclosed Pgp3 lateral flow assay (LFA-cassette) and a pared-back "dipstick" assay (LFA-dipstick). DBS were also tested with a bead-based multiplex assay (MBA). There was no significant difference in antibody positivity between the MBA and either LFA format (ranging from 42.5% to 48.4%). Interrater agreement between an expert rater and 3 different raters in field and lab settings was uniformly good, with Cohen's kappa >0.81 in all cases. Highlights • Antibodies to the Chlamydia trachomatis antigen Pgp3 can be detected on a lateral flow test with high reproducibility. • The rapid lateral flow test measures antibodies to Pgp3 similar to highly sensitive bead-based assays. • The Pgp3 lateral flow assay has been modified to accommodate the use of dried blood spots, facilitating its use in laboratories. • Laboratory and field staff can be easily trained to read the Pgp3 lateral flow assay with good interrater agreement. [ABSTRACT FROM AUTHOR]