Urinary tract infections (UTIs) currently rank among the most prevalent infectious diseases worldwide, with chronic and recurrent infections being especially problematic. Uropathogenic E. coli (UPEC), which is a causative agent in most cases of UTIs, expresses a multitude of virulence factors. Some virulence factors and specific genes were examined by PCR method. Genetic diversity was evaluated by phylogenetic typing groups. To know the genetic linkages among various E. coli, we evaluated clonal relatedness among different sources of E. coli isolates in Iranian children with UTI and age-matched healthy people by PFGE. Some pathogenicity determinants were more prevalent in urinary strains rather than fecal E. coli strains, significantly. There were PAI IICFT073, PAI II J96, PAI I536, PAI ICFT073, PAIII536, PAI IV, gafD, focG, vat, usp, hlyD, sat, cnf1, picU, fliC (H7), kpsMTII, kpsMTIII. UPEC were mainly found in phylogenetic typing groups B2 and D, while in fecal isolates, phylogenetic groups A and D were the most common. One hundred and fifty E. coli genomic DNA were digested by XbaI restriction enzyme and subjected to PFGE. A high level genetic diversity of DNA restriction pattern was reported. So, different clonalities were observed among different sources of E. coli isolates in Iranian children with UTI and age-matched healthy people. With attention to these results, genetic patterns showed that the strains had different clonalities. The differences in the PFGE pattern are a result of various genetic events caused by consistent changes with some independent genetic events. Lack of genetic similarity among E. coli isolates with different sources can be explained by the acquisition of different gene factors.