Background: Urinary tract infections (UTIs) are among the most prevalent infections in hospitals and communities worldwide. Objectives: Due to the medical importance of UTIs caused by uropathogenic Escherichia coli (UPEC), this study aimed to investigate pathogenicity island (PAI) markers, O-antigen serogroups, and resistance to antibiotic agents associated with UPEC isolates obtained from hospitalized patients in Rasht city hospitals. Methods: A total of 110 urine samples were taken from patients with UTI referred to selected hospitals in Rasht, Iran. The double-disk synergy test (DDST) was used to detect the isolate’s ability to produce extended-spectrum β-lactamase (ESBL). Using particular primers, eight PAIs were detected (ie, PAI I536, PAI II536, PAI III536, PAI IV536, PAI ICFT073, PAI IICFT073, PAI IJ96, and PAI IIJ96). Results: According to the antibiotic susceptibility pattern, a high level of antibiotic resistance was observed against nalidixic acid (81.8%) and co-trimoxazole (78.2%), while the most effective agent was amikacin (85.5%). Double-disk synergy test revealed that the incidence of ESBL-positive strains was 62.7% (69/110). Of the 110 UPEC isolates, 106 (96.4%) carried at least one of the investigated PAI markers. UPEC isolates with PAI IV536 (81.8%) had the highest prevalence, and PAI J196 (6.4%) had the lowest PAI marker. The most predominant serogroup O was O25 (36.4%), followed by O16 (17.3%), while the O4 and O7 serogroups (0.9%) were the lowest serogroups among UPEC isolates. Conclusions: The characterization of our strain revealed the co-occurrence of PAI and serogroups, confirming the importance of antibiotic resistance among the distinct serogroups and PAI markers. Our results have potential application for epidemiological studies and designing UTI treatment strategies against UTIs caused by UPEC.