Antimicrobial resistance (AMR) to gonorrhoea is a threat to global health security. There have been concerns expressed that countries with high rates of disease have poor surveillance. The objectives of the study were to determine the AMR patterns of Neisseria gonorrhoeae clinical isolates to antimicrobial agents in patients with HIV or high risk of HIV acquisition, to compare the concordance of disk diffusion and agar dilution as methods for determining AMR to N. gonorrhoeae, and to describe methodological challenges to carrying out AMR testing. The study was conducted at an HIV outpatient service for at-risk populations and an outreach clinic for commercial sex workers in Kampala. Patients were offered a sexually transmitted infection screen using a polymerase chain reaction (PCR)-based assay. Samples positive for gonorrhoea were cultured. Antimicrobial susceptibility testing was performed using disk diffusion and isolates were sent to a reference laboratory for agar dilution direct susceptibility testing. Five hundred and seventy-five patients were screened. There were 33 (5.7%) patients with gonorrhoea detected by PCR. Of the 16 viable N. gonorrhoeae isolates, 100% were resistant to ciprofloxacin and tetracycline by disk diffusion and 31% exhibited reduced susceptibility to ceftriaxone and cefixime. By agar dilution, 100% of isolates were resistant to ciprofloxacin and all isolates were susceptible to ceftriaxone and cefixime. There was concordance between disk diffusion and agar dilution for ciprofloxacin and tetracycline resistance and a significant discordance for third-generation cephalosporins. More than half the women with gonorrhoea were asymptomatic and represent a potential reservoir for ongoing transmission. AMR testing of N. gonorrhoeae isolates is needed to ensure optimal treatment and prevention of antibiotic resistance progression.