Background: Urinary Tract Infections (UTIs) are the most common infectious diseases in childhood. The Uropathogenic Escherichia coli (UPEC) strains account for as much as 80% of UTIs. Objective: From a clinical perspective, it is important to know which virulence factors and antibiotic resistance properties are present in UPEC strains in pediatrics. Therefore, this study was carried out to investigate the molecular characterization and antimicrobial resistance of UPEC strains isolated from hospitalized patients in pediatric ward of Baqiyatallah Hospital in Tehran. Patients and Methods: One hundred and twenty-one urine specimens were collected from the patients infected with UTIs (51 boys and 70 girls). The urine samples were cultured immediately, and those with E. coli-positive were analyzed for the presence of antibiotic resistance genes and bacterial virulence factors using Polymerase Chain Reaction (PCR). Also, antimicrobial susceptibility testing was performed using disk diffusion methodology with Mueller-Hinton agar according to the instruction of Clinical Laboratory and Standard Institute. Results: Nineteen out of 51 (37.25%) urine samples from boys and 47 out of 70 (67.14%) urine samples from girls harbored E. coli. A significant difference was found between the frequency of UPEC strains in boys and girls (P <.05). High resistance levels to tetracycline (69.6%), ampicillin (69.6%) and norfloxacin (63.6%) were also observed. Totally, 1.66% of tested strains were resistant to more than 8 antibiotics. The incidence of genes encoding resistance against gentamicin (aac (3)-IV), sulfonamide (sul1), beta-lactams (blaSHV and CITM), tetracycline (tetA and tetB), trimethoprim (dfrA1), and quinolones (qnr) were 25. 7%, 22.7%, 83.2%, 71.1%, 19.6% and 21.2%, respectively. The most commonly detected virulence factors were fim (71.2%), set-1 (66.6%), iha (62.1), papGI (59%), usp (56%) and sen (22.7%). Conclusion: Resistant strains of uropathogenic E. coli had the lower incidence of uropathogenic virulence factors. We suggested prescription of imipenem and amikacin to treat pediatric patients infected with UTIs.