Abstract: Objective To analyze the distribution of pathogenic bacteria in urine samples of patients with urinary tract infections in the Affiliated Hospital of Xuzhou Medical University and the resistance of Escherichia coli to commonly used antibiotics, so as to provide guidance for the rational selection of antibiotics.Methods A total of 3 882 self-collected or sterile urine samples were collected in the Affiliated Hospital of Xuzhou Medical University from August 2021 to July 2022. The midstream urine culture was carried out to analyze the distribution of pathogens in urine, the department distribution and antimicrobial susceptibility of Escherichia coli.Results A total of 881 (22.69%) in 3 882 cultured urine samples were positive for bacteria, where 846 were caused by a single strain and 35 were caused by two mixed strains; 916 strains of pathogenic bacteria were isolated, including 512 strains of gram-negative bacteria (55.90%), where Escherichia coli (306 strains, 33.41%) covered the largest percentage, followed by Klebsiella pneumoniae (75 strains, 8.19%);188 strains of gram-positive bacteria (20.52%), where Enterococcus faecium (80 strains, 8.73%) covered the largest percentage, followed by Enterococcus faecalis (27 strains, 2.95%); 216 strains of fungi (23.58%), where Candida albicans (77 strains, 8.41%) and Candida tropicalis (58 strains, 6.33%)covered the largest percentage. Among the 306 strains of uropathogenic Escherichia coli (UPEC), 8.50% (26/306) were isolated from outpatients and 91.50% (280/306) from inpatients, with the highest detection rates of patients in Departments of Urology, Nephrology, Rheumatology, Neurology and intensive care unit(ICU), which accounted for 32.68%, 20.92%, 8.50%, 6.86% and 6.21%, respectively. The resistance of UPEC to ticarcillin (88.24%), cefuroxime (61.49%), cefuroxime axetil (62.94%), levofloxacin (72.46%) and compound sulfamethoxazole (58.03%) were higher than 50%; but sensitive to other antibacterial drugs.Conclusions The composition of pathogens in this hospital is complicated and varied in the positive specimens of patients with urinary tract infections. Gram-negative bacteria are the leading cause, with Escherichia coli accounting for the largest percentage. Parts of pathogens present high levels of drug resistance. Patients should actively cooperated with physicians to carry out pathogen identification and antimicrobial susceptibility test, so that to select antibiotics reasonably for the treatment of urinary tract infections, and effectively avoid the emergence of multi-drug resistant bacteria and new drug-resistant bacteria.