The number of resistant strains in patients with Neisseria gonorrhoeae urethritis has been increasing, making effective treatment difficult. Chromosomally mediated penicillin-resistant N. gonorrhoeae arise through alterations in penicillin-binding proteins (PBPs) and a decrease in outer membrane permeability. To understand the occurrence of penicillin resistance in patients with N. gonorrhoeae infection, we performed this study. In addition, we studied minimum inhibitory concentrations (MICs) of antimicrobials against N. gonorrhoeae strains. We measured the MICs of penicillin G, other beta-lactams, and other kinds of antimicrobials against 53 clinical N. gonorrhoeae isolates from male patients with urethritis in Hyogo and Osaka, Japan. The ponA genes, encoding PBP 1 of these isolates, were sequenced. Of the 53 isolates tested, 41 strains showed some resistance to penicillin G. A mutation in the ponA (ponA1) gene was identified in 46 isolates. There was a tendency that ponA mutant (ponA1) in N. gonorrhoeae led to higher antimicrobial MICs of beta-lactam antimicrobial agents (including penicillin) than those of non-ponA mutants. However, we found lower than expected MICs of penicillin and beta-lactams even in ponA mutants. Therefore, we consider that detailed investigations for the further understanding of the effect of other genes, such as penC (which is reported to be related to ponA1 in achieving high-level penicillin resistance) should be our next step.