Background. Molecular typing was used to elucidate Neisseria gonorrhoeae transmission networks among men who have sex with men (MSM) in Amsterdam, the Netherlands. We determined whether clusters of patients infected with specific N. gonorrhoeae genotypes were related to various epidemiological characteristics. Methods. MSM (age ≥18 years) visiting the sexually transmitted infections (STI) clinic between July 2008 and August 2009 were eligible. After STI screening, participants completed a behavioral questionnaire concerning the previous 6 months. N. gonorrhoeae cultures were genotyped using multiple-locus variable-number tandem repeat analysis typing. Results. We obtained 278 N. gonorrhoeae– positive isolates from 240 MSM. Five large clusters (≥10 isolates), a unique sixth cluster ( n = 9), and 8 smaller clusters (5–9 isolates) were identified. Prevalence of human immunodeficiency virus differed between clusters I and VI ( P = .003), ranging from 27.8% to 100%. Receptive unprotected anal intercourse was frequently reported by MSM (51.8%) but did not differ significantly among clusters. Significant differences were identified concerning the participant's history of syphilis ( P = .030), having met partners at a popular sex venue in Amsterdam ( P = .048), and meeting partners outside Amsterdam ( P = .036). Conclusions. Distinct N. gonorrhoeae transmission networks were present in a mixed high-risk MSM population; concordance between clusters and epidemiological characteristics was present but not marked.