<jats:title>ABSTRACT</jats:title> <jats:p>Empirical gonorrhea treatment at initial diagnosis reduces onward transmission. However, increasing resistance to multiple antibiotics may necessitate waiting for culture-based diagnostics to select an effective treatment. There is a need for same-day culture-free diagnostics that identify infection and detect antimicrobial resistance. We investigated if Nanopore sequencing can detect sufficient <jats:named-content content-type="genus-species">Neisseria gonorrhoeae</jats:named-content> DNA to reconstruct whole genomes directly from urine samples. We used <jats:named-content content-type="genus-species">N. gonorrhoeae</jats:named-content>-spiked urine samples and samples from gonorrhea infections to determine optimal DNA extraction methods that maximize the amount of <jats:named-content content-type="genus-species">N. gonorrhoeae</jats:named-content> DNA sequenced while minimizing contaminating host DNA. In simulated infections, the Qiagen UCP pathogen mini kit provided the highest ratio of <jats:named-content content-type="genus-species">N. gonorrhoeae</jats:named-content> to human DNA and the most consistent results. Depletion of human DNA with saponin increased <jats:named-content content-type="genus-species">N. gonorrhoeae</jats:named-content> yields in simulated infections but decreased yields in clinical samples. In 10 urine samples from men with symptomatic urethral gonorrhea, ≥92.8% coverage of an <jats:named-content content-type="genus-species">N. gonorrhoeae</jats:named-content> reference genome was achieved in all samples, with ≥93.8% coverage breath at ≥10-fold depth in 7 (70%) samples. In simulated infections, if ≥10<jats:sup>4</jats:sup> CFU/ml of <jats:named-content content-type="genus-species">N. gonorrhoeae</jats:named-content> was present, sequencing of the large majority of the genome was frequently achieved. <jats:named-content content-type="genus-species">N. gonorrhoeae</jats:named-content> could also be detected from urine in cobas PCR medium tubes and from urethral swabs and in the presence of simulated <jats:italic>Chlamydia</jats:italic> coinfection. Using Nanopore sequencing of urine samples from men with urethral gonorrhea, sufficient data can be obtained to reconstruct whole genomes in the majority of samples without the need for culture.</jats:p>