The percentage of quinolone-resistant Neisseria gonorrhoeae isolated in London increased between 2000 and 2003, from 0.9% to 7.9% of total isolates. This increase was investigated by genotyping resistant isolates and comparing demographic and behavioral data. In 2000, resistant isolates predominantly had unique sequence types (STs) that were associated with imported infection, whereas, in 2002 and 2003, large ST clusters of indistinguishable isolates were associated with endemic acquisition. Resistant isolates that belonged to these large clusters were typically from patients who had similar epidemiological characteristics (such as ethnicity and sexual orientation) and behavioral characteristics (such as multiple sex partners and previous gonorrhea). In London, quinolone resistance is no longer associated with importation from areas of high prevalence and is spreading endemically in high-risk groups.