Introduction: Healthcare-associated urinary tract infection (UTI) represents a significant health problem, especially in infants and young children. The most common pathogen associated with this infection is Escherichia coli (E.coli). Objective: The present study aimed to detect the frequency of virulence genes among clinical isolates of E.coli isolated from healthcare-associated urinary tract infections in children and the correlation between these virulence genes and the presence of the bla-CTX gene. Methods: The study included one hundred clinical isolates of E.coli isolated from healthcare-associated urinary tract infections in children in intensive care units. The isolates were subjected to antibiotics sensitivity by disc diffusion method and detection of extended-spectrum beta-lactamase by double disc diffusion method. In addition, multiplex polymerase chain reaction (PCR) was used to detect some virulence genes, and PCR was used to detect the bla-ctx-M gene. Results: E.coli producing ESBL by double discs method was identified in 74 isolates. bla-ctx-M gene detection by PCR was identified among 38 isolates representing 51.4% of ESBL-producing E.coli. There was a significant association between ESBL and bla-ctx-M Gene, P=0.0001. The frequency of the studied virulence genes by multiplex PCR in the isolated E.coli was 66% for the Fim gene, 75% for the Aer gene, 68% for the FliC gene, 53% for each of IucD gene and Usp gene, 40% for pap gene, 35% for each of AFA and ironN genes and 17% for sfa gene. None of the isolated E.coli had the Cdt gene. There was a significant association between the presence of the FimH gene (P=0.0001), Pap gene (P=0.05), sfa (P=0.026), Afa gene (P=0.018), and aer gene (P=0.035) and the presence of the bla-ctx-M gene in the isolated E.coli. Conclusion: The present study highlights the presence of virulence genes and bla-ctx-M gene in uropathogenic E.coli isolated from pediatric patients with healthcare-associated urinary tract infections. There was an association between the bla-ctx-M gene and virulence genes FimH, pap, sfa, Afa, and aer. Various distributions of the studied genes with a high frequency of fimbria are flic genes. Moreover, the ESBL had high frequency in E.coli with the presence of bla-ctx-M in about one-third of the isolates.