Introduction: Urinary tract infection (UTI) is one of the most common bacterial infections in childhood, and the increasing rate of antibiotic resistance to the commonly prescribed antimicrobial agents against it has become a major concern. The aim of this study was to determine the antibiotic resistance and genotyping of bacteria isolated from urine cultures in patients referred to the Children's Medical Center, Tehran, Iran. Methods: During the 1-year period, antimicrobial susceptibility profiles of strains isolated from patients with UTI were determined. Typing of the isolates causing nosocomial infections was performed by random amplified polymorphic DNA (RAPD) analysis, and the results were analyzed by Gelcompar II software. Results: In this study, 203 children (130 girls and 73 boys) were included. The patients' age ranged from 1 day to 16 years (IQR average=4 months to 4 years). The most frequent isolated organisms were Escherichia coli (118 isolates, 58%), followed by Klebsiella pneumoniae (30 isolates, 15%). Sixty-two strains (18 strains of E. coli, 13 strains of K. pneumoniae, 11 strains of Enterococcus faecium, and five strains of Burkholderia cepacia complex) had criteria of nosocomial infection. A high resistance rate to trimethoprim-sulfamethoxazole (69%) and cefotaxime (60%) was reported in E. coli and K. pneumoniae strains, respectively. Pseudomonas aeruginosa strains showed high sensitivity to amikacin (100%). All E. faecium strains were susceptible to trimethoprim-sulfamethoxazole (100%), and 23% of the strains were resistant to vancomycin. The analysis of RAPD-typing revealed the presence of three clusters in E. coli, two clusters in E. faecium, and one clone in K. pneumoniae. Besides, four out of five isolates of B. cepacia complex had more than 90% genetic similarity. Conclusion: The most frequent isolated pathogen was E. coli, and an increasing rate of antibiotic resistance to the commonly prescribed antimicrobial agents such as trimethoprim/ sulfamethoxazole and cephalosporins was observed. Moreover, the results of this study showed the presence of clones with â„80% similarity in E. coli, K. pneumoniae, E. faecium, and B. cepacia complex isolates; therefore, the transmission of nosocomial infections from one patient to another or one ward to another is probable.