Distancing measures during the COVID-19 lockdown led to a temporary decrease of casual sex partners among clients of the Centre for Sexual Health (CSH) in Amsterdam, the Netherlands. We investigated the effect of this change on the genotypic and phenotypic distribution ofNeisseria gonorrhoeae(Ng) isolates from CSH patients. From eachNg-positive patient we sequenced one isolate, resulting in 322 isolates which constituted two groups: 181 isolates cultured from 15 January to 29 February 2020 (before the first lockdown) and 141 cultured from 15 May to 30 June 2020 (during the first lockdown). Patient characteristics showed significantly more symptomatic patients and significantly fewer reported sex partners during the lockdown. Phenotypic data showed an increase in low-level azithromycin resistance and ceftriaxone susceptibility during the lockdown, and this remained after the study period. The diversity in sequence types (STs) decreased slightly during the lockdown. A shift occurred from ST 8156 being predominant before lockdown to ST 9362 during lockdown and a remarkably low median SNP distance of 17 SNPs was found between ST 9362 isolates obtained during lockdown. These findings reflect restricted travel and the change in sexual behaviour of CSH clients during the lockdown, with a potentially increased local transmission of the ST 9362 strain during this period, which led to genotypic and phenotypic changes in theNgpopulation. This shows that public health measures have far-reaching consequences and should be considered in the surveillance of other infectious diseases.