Abstract Neisseria gonorrhoeae is the sole causative agent of the sexually transmitted infection gonorrhea that is restricted to the human population. N. gonorrhoeae has co-evolved with its human host throughout all modern history and has evolved specific mechanisms of pathogenesis that relate to its mode of transmission and the anatomical sites it colonizes. Rates of gonorrhea are rising along with a rapid acquisition of antibiotic resistance. There is an urgent need to understand of the mechanisms of N. gonorrhoeae pathogenesis to develop new treatment options or vaccine candidates. N. gonorrhoeae is naturally competent for DNA transformation and is one of a few bacterial species that does not regulate competence. This resource grant will leverage the transformation competence of N. gonorrhoeae and synthetic DNA constructs to create ordered gene knockout libraries of the two most commonly used N. gonorrhoeae strains, MS11 and FA1090. These strains represent different phylogenetic clades of the bacterium. The libraries will include deletions of all genes encoding an open reading frame (ORF) that will allow the replacement of the ORF with a non-polar kanamycin resistance gene (KmR). Each KmR insertion mutant will also carry a unique random barcode. Leading researchers in the field and NIAID program officials also support this resource proposal as an important shared resource to help advance progress in this field.