Background: Escherichia coli is the leading causative agent of urinary tract infections worldwide and represents a major public health concern due to its increasing resistance to commonly used antimicrobial agents. Rapid molecular identification, together with the characterization of antimicrobial resistance profiles, is essential for effective patient management and infection control. Objective: This study aimed to molecularly identify E. coli isolates obtained from urine cultures at Shamiya State Hospital, Iraq, and to determine their antimicrobial resistance patterns to support appropriate therapeutic strategies. Methods: Urine samples were collected from patients attending Shamiya State Hospital, Iraq, and processed using standard microbiological procedures. Bacterial isolates presumptively identified as Escherichia coli were obtained through culture on selective and differential media and confirmed by conventional biochemical tests. Molecular identification was performed by polymerase chain reaction (PCR) targeting species-specific genetic markers. Antimicrobial susceptibility testing was carried out using the disk diffusion method in accordance with Clinical and Laboratory Standards Institute (CLSI) guidelines. Results: The isolates comprised 29% E. coli, 21% Staphylococcus aureus, 18% Klebsiella pneumoniae, 12% Proteus mirabilis, 16% Enterococcus faecalis, and 4% Pseudomonas aeruginosa. Among the resistance genes, the CTX-M gene exhibited the strongest resistance profile, while the TET-A and TEM genes showed weaker resistance. The QNR-B gene demonstrated the weakest resistance compared to the CTX-M gene among all isolates. In addition, the demographic characteristics of the individuals from whom the E. coli strains were isolated were recorded. The results obtained contributed to the development of clinical strategies for the prevention and management of uropathogenic E. coli (UPEC) infections in the Shamiya region.