Mutations in the peptidyltransferase loop in domain V of 23S rRNA alleles have been associated with macrolide resistance in Neisseria gonorrhoeae. This study analyzed three Argentinian gonococcal strains with different azithromycin susceptibilities (high-level azithromycin-resistant [AzHLR] strain 1782, MIC 2048 mg/liter; resistant strain 2498, MIC 8 mg/liter; susceptible strain 3783, MIC 0.06 mg/liter) using a two-step PCR method, with sequences compared to known azithromycin-resistant strains. The susceptible strain showed no mutations in the 23S rRNA rrl gene. Strain 2498 had the C2599T mutation in all four rrl alleles and a TT insertion in the mtrR promoter region, representing the first clinical isolate with both resistance mechanisms. Strain 1782 harbored an A2143G mutation (corresponding to Escherichia coli A2059G, not previously reported in gonococci) in all four rrl alleles, which is strongly suggested to be responsible for its high-level azithromycin resistance. These findings illustrate how gonococcal strains adapt under antibiotic selection pressure.