Antimicrobial resistance (AMR) poses a significant global health threat, particularly due to increasing bacterial resistance to β-lactam and aminoglycoside antibiotics, primarily mediated by extended-spectrum β-lactamases (ESBLs) and 16S rRNA methylases in Enterobacteriaceae. In this study, a multidrug resistant (MDR) E. coli strain HN257 isolated from chicken belonging to ST224 and serotype O88:H23 was characterized. SNP-based phylogenetic analysis revealed two distinct clades among poultry-associated E. coli ST224 in this study and others from Genbank, with strain HN257 closely related to chicken-derived E. coli YH17148 (serotype O78:H23), from China. The E. coli HN257 harbored four plasmids with 16 resistance determinants. Two bla