Background: Uropathogenic Escherichia coli (UPEC) is the most common cause of urinary tract infection (UTI) in both hospital and community settings. The recent emergence of multidrug-resistant Escherichia coli (E. coli) poses an alarming threat to health. The development of a novel antibiotic against resistant UPEC is a pressing need. Identification of virulent and resistant organisms is essential to target them specifically. Materials and methods: In this study, an established multiplex polymerase chain reaction assay was used to detect 29 virulence factor (VF) genes among 50 UPEC strains collected from patients from Saudi Arabia, and associations between virulence profiles and sequence types were observed. Results: fimH (mannose-specific adhesin of type 1 fimbriae: overall frequency, 92%), traT (serum resistance, 84%), fyuA (yersiniabactin, 64%), iutA (aerobactin, 64%), kpsMT II (capsule group II, 56%), and PAI (pathogenicity island marker, 48%) were the most frequent VF genes among 50 UPEC isolates. bmaE (M fimbriae), gafD (G fimbriae), nfaE (non-fimbrial adhesin), cdtB (cytolethal distending toxin), and kpsMT III (capsule group III) were not found in any strains tested. The most common sequence types were ST131 (14%), ST69 (10%), ST38 (10%), ST95 (8%), ST10 (8%), ST73 (8%), and ST127 (6%). Some isolates of ST127 and ST73 carried the highest number of VF genes (14), whereas ST131 isolates expressed fewer. Conclusion: These findings may help detect virulent E. coli strains and assess their resistance potential, with the aim of developing antibiotics targeting the most prevalent VF genes to treat resistant E. coli.