A prospective multicenter study was conducted to assess the epidemiology of antimicrobial resistance among uropathogens causing uncomplicated cystitis. Adult women with clinical diagnosis of uncomplicated cystitis were enrolled from 54 participating centers distributed all over Hong Kong during 2006 to 2008. A positive urine culture was found in 59.5% (352/592) patients. The patients had mean age of 44.9 years, and most (89.2%) were otherwise healthy. The most prevalent causative organism was Escherichia coli (77%), followed by other Enterobacteriaceae (14.2%), staphylococci (5.1%), and other Gram-positive bacteria (3.7%). The resistance rates of E. coli to co-trimoxazole and ciprofloxacin were 29.5% and 12.9%, respectively, and 14 isolates (5.2%) were confirmed as extended-spectrum beta-lactamase (ESBL) producers. Of the ESBL producers, molecular studies showed CTX-M-14, CTX-M-24, or CTX-M-9. Nitrofurantoin and fosfomycin were active against >90% of the isolates, regardless of resistance phenotypes for other drugs. Pulsed-field gel electrophoresis of representative isolates showed that the antibiotic-resistant strains were genetically diverse. Patients with history of recent antibiotic use were significantly more likely to have infection by E. coli with co-trimoxazole resistance (odds ratio [OR], 2.8; 95% confidence interval [CI], 1.4-5.7; P = 0.003) and ciprofloxacin resistance (OR, 2.5; 95% CI, 1.1-5.8; P = 0.03). Knowledge of the resistance data and risk factors could inform better use of antibiotics for empiric therapy for acute uncomplicated cystitis.