BACKGROUND: Performance of a 16S rRNA analysis of the cervicovaginal microbiome of 220 participants recruited into the T Cell Response against Chlamydia (TRAC) cohort between February 2011 and August 2014 in Allegheny County, Pennsylvania USA detected DNA encoding chlamydial 16S rRNA in samples from seven participants whose tests were negative for Chlamydia trachomatis (CT) and DNA encoding gonococcal 16S rRNA from five participants whose tests were negative for Neisseria gonorrhoeae (NG) infection with the Aptima Combo 2 assay (Hologic). METHODS: We used targeted PCR amplification followed by sequencing to characterize the chlamydial 23S rRNA locus and qPCR to detect gonococcal DNA in residual diagnostic swab eluates or DNA used to generate 16S rRNA libraries. RESULTS: Discrepant specimens that contained chlamydial DNA carried a diagnostic-avoidant, G1523A nucleotide polymorphism in the 23S rRNA locus identical to variants previously detected in Finland, Denmark, and the UK. PCR validation of gonococcal DNA was confirmed for all participants whose tests were negative, with stochastic effects consistent with infection levels close to the limit of detection by the diagnostic assay. CONCLUSIONS: These data indicate that this probe-avoidant CT mutant was circulating in the northeastern US prior to its detection and characterization in 2019. Although infrequent, false negative tests could represent infection with probe-avoidant CT mutants. Additional research is needed to determine if there is a role for CT tests using alternate probes for symptomatic or exposed individuals suspected of infection with these mutant chlamydial strains.