<jats:sec id="sec001"><jats:title>Background</jats:title><jats:p>The increasing rate of antimicrobial-resistant<jats:italic>Neisseria gonorrhoeae</jats:italic>poses a considerable public health threat due to the difficulty in treating gonococcal infections. This study examined antimicrobial resistance (AMR) to drugs recommended for gonorrhea treatment between 2015 and 2017, and the AMR determinants and genetic compositions of plasmids in 3 gonococcal strains with high-level penicillin resistance.</jats:p></jats:sec><jats:sec id="sec002"><jats:title>Methods</jats:title><jats:p>We collected 117<jats:italic>N</jats:italic>.<jats:italic>gonorrhoeae</jats:italic>isolates from patients with gonococcal infections who attended Siriraj Hospital, Bangkok, Thailand, between 2015 and 2017. Minimum inhibitory concentrations (MICs) of penicillin, tetracycline, ciprofloxacin, azithromycin, spectinomycin, cefixime, and ceftriaxone were determined by the agar dilution method. PCR amplification and sequencing of 23S rRNA and<jats:italic>mtrR</jats:italic>(a negative regulator of MtrCDE efflux pump) were performed. Whole genomes of 3 PPNG strains with high-level penicillin resistance (MIC ≥ 128 μg/ml) were sequenced using Illumina and Nanopore sequencing platforms.</jats:p></jats:sec><jats:sec id="sec003"><jats:title>Results</jats:title><jats:p>The proportions of<jats:italic>N</jats:italic>.<jats:italic>gonorrhoeae</jats:italic>isolates with resistance were 84.6% for penicillin, 91.5% for tetracycline, and 96.6% for ciprofloxacin. All isolates were susceptible to spectinomycin, azithromycin, cefixime, and ceftriaxone. An adenine deletion within a 13 bp inverted repeat sequence in the<jats:italic>mtrR</jats:italic>promoter and an H105Y mutation in the<jats:italic>mtrR</jats:italic>coding region were found in the<jats:italic>N</jats:italic>.<jats:italic>gonorrhoeae</jats:italic>isolate with the highest azithromycin MIC value (1 μg/ml). Three high-level penicillin-resistant isolates contained nonmosaic type II<jats:italic>penA</jats:italic>and had mutations in<jats:italic>penB</jats:italic>and the<jats:italic>mtrR</jats:italic>coding region. All isolates with high-level penicillin resistance carried the conjugative plasmids with or without the Dutch type<jats:italic>tetM</jats:italic>determinant, the beta-lactamase plasmid (Rio/Toronto), and the cryptic plasmid.</jats:p></jats:sec><jats:sec id="sec004"><jats:title>Conclusions</jats:title><jats:p>The gonococcal population in Thailand showed high susceptibility to ceftriaxone and azithromycin, current dual therapy recommended for gonorrhea treatment. As elevated MIC of azithromycin has been observed in 1 strain of<jats:italic>N</jats:italic>.<jats:italic>gonorrhoeae</jats:italic>, expanded and enhanced surveillance of antimicrobial susceptibility and study of genetic resistance determinants are essential to improve treatment guidelines.</jats:p></jats:sec>