Aim: To characterize mutations in penA, mtrR, ponA, and porBIB, considered target genes for antimicrobial resistance, in Neisseria gonorrhoeae isolates with elevated minimum inhibitory concentrations (MICs) of ceftriaxone cultured from patients in New Zealand. Results: Out of 28 isolates supplied by the Institute of Environmental Science and Research Limited (ESR), Porirua, New Zealand, 14 were found to show reduced susceptibility to ceftriaxone (MIC of 0.06 mg/L) according to criteria used by the ESR and the Australian Gonococcal Surveillance Programme (AGSP) when tested in our laboratory. Rates of resistance to ciprofloxacin, azithromycin, penicillin, and tetracycline were 100% (28/28), 7% (2/28), 36% (10/28), and 25% (7/28), respectively. Ten different penA (Penicillin binding protein 2 [PBP2]) sequences were observed. The most common mosaic penA M-1 resembled mosaic penA XXXIV, which has been associated with ceftriaxone treatment failures in other countries. Four semimosaic PBP2 sequences were observed and may be novel PBP sequences, while four out of five nonmosaic PBP2 sequences were similar to PBP2 sequences reported in Australia. Twenty-one isolates harbored mutations in all 4 genes (penA, mtrR, porBIB, and ponA), and 13 of these exhibited reduced susceptibility to ceftriaxone. Conclusion: Mutations in penA, mtrR, porBIB, and ponA observed in this study may have contributed to reduced susceptibility to ceftriaxone among New Zealand gonococcal isolates. Over half (16/22) of mosaic penA sequences from the gonococcal isolates resembled penA XXXIV.