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ABSTRACT The strict human pathogenNeisseria gonorrhoeae(gonococcus [Gc]) infects an estimated 82 million individuals globally and is a World Health Organization-designated bacterial pathogen of public health importance due to escalating antimicrobial resistance. Gc vaccines have been hindered by Gc’s ability to evade immune surveillance in part by varying its major surface antigens like the type IV pilus. We developed a quick and precise method for measuring pilin antigenic variation (Av) frequency using droplet digital PCR (ddPCR) technology. Two fluorescent probes were designed to detect either the hypervariable tail region of silent pilin locuspilS3-copy 1 (S3C1) or a sequence conserved in allpilEvariants (CYS2). The appropriate frequency of pilin antigenic variation is measured by the proportion ofpilEamplicons carrying the recombinant S3C1 copy relative to the total pilE amplicons measured by CYS2. The ddPCR assay is specific for RecA-dependent pilin antigenic variation. The reduced frequency of pilin Av in strains lacking RecA-modulating recombination protein RecX and the DNA helicase RecQ confirms the ability of the assay to measure changes in pilin Av frequency. We used the ddPCR assay to determine that pilin Av frequency is altered by the colony densities on a solid medium. The ddPCR assay is an accurate, efficient way to measure Gc pilin Av frequency. IMPORTANCEGonorrhea is a sexually transmitted infectious disease of the human genital and nasopharyngeal mucosa caused by the host-restricted bacteriumNeisseria gonorrhoeae. The rise of antibiotic-resistant gonorrhea is an urgent global threat to public health. Pilus antigenic variation is a gene conversion process that allowsN. gonorrhoeaeto evade host immune surveillance, and a mechanistic understanding of this process is crucial to understandingN. gonorrhoeaepathogenesis. This report shows that we can adopt a digital PCR methodology to quickly and accurately measure pilin antigenic variation.