The goal of this work was to determine the factors affecting the emergence of azithromycin-resistantNeisseria gonorrhoeaeisolates in Russia, where azithromycin was never recommended for the treatment of gonococcal infections.Clinical N. gonorrhoeaeisolates collected in 2018–2021 (428 isolates) were analyzed. No azithromycin-resistant isolates were found in 2018–2019, but in 2020–2021, a significant increase in the ratio of azithromycin-resistant isolates was observed: 16.8% and 9.3%, respectively. A hydrogel DNA microarray was developed for the analysis of resistance determinants: mutations in the genes encoding the mtrCDE efflux system and in all four copies of the 23S rRNA gene (position 2611). A majority of the azithromycin-resistant Russian isolates belonged to the NG-MAST G12302 genogroup, and the resistance was associated with the presence of a mosaic structure of themtrRgene promoter region with the −35 delA deletion, an Ala86Thr mutation in themtrRgene, and a mosaic structure of themtrDgene. A comparative phylogenetic study of modern Russian and EuropeanN. gonorrhoeaepopulations allowed us to conclude that the emergence of azithromycin resistance in Russia in 2020 was the result of the appearance and spread of EuropeanN. gonorrhoeaestrains belonging to the G12302 genogroup due to possible cross-border transfer.