Antibiotic resistance accounts for over 50% of Escherichia coli (E. coli) infections, mediated by extended-spectrum β-lactamases (ESBLs), AmpC β-lactamases, carbapenemase, and other mechanisms. Data on AmpC, carbapenemase, aminoglycosides, and quinolones resistance of E. coli in Jordan are minimal. This study aimed to determine the molecular and phenotypic prevalence of antibiotic-resistant E. coli in Jordan. Methods: 153 E. coli isolates collected from multiple Jordanian hospitals were tested for species identification, antibiotic susceptibility, and resistance genes. Results: 153 E. coli isolates were collected with a mean age of 47.09 ± 25.32. Most samples were collected from the emergency department (29.7%) and urine samples were the major source (82.9%). For AmpC detection, 67 (57.8%) samples were resistant to cefoxitin, 13 (23.2%) were positive for AmpC disk test, all tested samples were negative for CMY-1 gene, while 15 (14.4%) samples were positive for CMY-2 gene. Regarding aminoglycoside resistance, 54 (38 %) strains were resistant to gentamycin, 3 (2.1%) were resistant to amikacin, and 94 (61.4%) samples had aac6’-Ib-cr gene. For fluoroquinolones resistance, 92 (65.7 %) isolates were resistant to ciprofloxacin, 65 (47.1%) were resistant to levofloxacin, and 102 (98%) isolates were positive for the gyrA gene. Finally, 3 (2%) isolates were resistant to imipenem and meropenem, however, carbapenemase genes including KPC, OXA-48, IMP, and VIM genes were negative in all samples. Conclusion: Understanding the molecular and phenotypic characteristics of antibiotic-resistant E. coli will help to guide proper antibiotic therapy and combat microbial resistance in Jordan.