Antimicrobial resistance is one of the major public health threats globally. This challenge has been aggravated with the overuse and misuse of antibiotics in food animals and humans. The present study aimed to investigate the prevalence of Extended-Spectrum β-lactamase (ESBL) genes inEscherichia coli(E.coli) isolated from broiler chickens in Kelantan, Malaysia. A total of 320 cloacal swabs were collected from farms in different districts of Kelantan and were analyzed using routine bacteriology, antimicrobial susceptibility test, and molecular techniques for further identification and characterization of ESBL encoding genes. Based on PCR detection for theE.colispecies-specificPhogene, 30.3% (97/320) of isolates were confirmed asE.coli, and 84.5% (82/97) of the isolates were positive for at least one ESBL gene. Majority of the isolates, 62.9% (61/97) were harboringblaCTX-Mfollowed by 45.4% (44/97) ofblaTEMgenes, while 16.5% (16/97) of the isolates were positive for bothmcr-1and ESBL genes. Overall, 93.8% (90/97) of theE.coliwere resistant to three or more antimicrobials; indicating that the isolates were multi-drug resistance. 90.7% of multiple antibiotic resistance (MAR) index value greater than 0.2, would also suggest the isolates were from high-risk sources of contamination. The MLST result shows that the isolates are widely diverse. Our findings provide insight into the alarmingly high distribution of antimicrobial resistant bacteria, mainly ESBL producingE.coliin apparently healthy chickens indicating the role of food animals in the emergence and spread of antimicrobial resistance, and the potential public health threats it may pose.