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The emergence of Neisseria gonorrhoeae strains with decreased susceptibility to cephalosporins represents a major concern globally. The aim of this study was to examine the phenotypic and molecular characteristics of N. gonorrhoeae isolates with decreased susceptibility to ceftriaxone and cefixime in Argentina.A total of 1987 isolates were collected during 2009 and 2013. The susceptibility to penicillin G, tetracycline, ciprofloxacin, cefixime, ceftriaxone, and azithromycin was determined using the agar dilution method. The major extended-spectrum cephalosporin resistance determinants (penA, mtrR, and porB1b) were sequenced in 42 N. gonorrhoeae isolates that showed decreased susceptibility to ceftriaxone (minimum inhibitory concentration [MIC], 0.06–0.125 mg/L) and cefixime (MIC, 0.125–0.25 mg/L). Genotyping by N. gonorrhoeae multiantigen sequence typing (NG-MAST) was performed.Between 2009 and 2013, there was a shift in the modal MICs for ceftriaxone. Among the 42 isolates exhibiting decreased susceptibility to ceftriaxone and cefixime, 95.2% were resistant to penicillin G, 95.2% to tetracycline, 97.6% to ciprofloxacin, and 33.3% to azithromycin. Thirty-five (83.3%) of the 42 isolates had a mosaic penA allele XXXIV, which has been previously associated with resistance to ceftriaxone and cefixime as well as treatment failures. The isolates that contained the mosaic penicillin-binding protein 2 (PBP2) XXXIV were associated with NG-MAST ST1407 or closely related genotypes.In Argentina, N. gonorrhoeae isolates with decreased susceptibility to cefixime and ceftriaxone have now emerged, mostly due to the introduction of the internationally spread multidrug-resistant NG-MAST ST1407.