Abstract Ceftiofur, a third-generation cephalosporin, is classified as a critically important antimicrobial in both human and veterinary medicine, but its use in poultry has raised public health concerns due to an increase in resistant pathogens. This study comprehensively investigated the distribution and characteristics of ceftiofur-resistant Escherichia coli ( E. coli ) across the production stages of three major integrated broiler operations in Korea. Although ceftiofur was only used via subcutaneous injection in day-old chicks at breeder hatcheries and not in other stages of integrated broiler production, ceftiofur-resistant E. coli were detected throughout all stages of integrated broiler production. The prevalence of ceftiofur-resistant E. coli isolates was generally greater in fluff and eggshells from breeder hatcheries (15.4%–47.4%) and commercial broiler hatcheries (31.6%–51.7%) than in environmental dust from broiler breeder farms (11.5%–38.5%) and commercial broiler farms (8.7%–28.9%). Notably, resistant E. coli in slaughterhouse carcasses correlated with a high prevalence in farm dust at the time of flock depletion. Compared with the susceptible isolates (0.0%–75.7%), the ceftiofur-resistant isolates (20.5%–100.0%) presented significantly greater coresistance to other antimicrobials ( p < 0.05). Among the 239 resistant isolates, 92.9% harboured at least one β-lactamase gene, predominantly bla CTX-M-55 , bla TEM-1 , or bla CMY-2 (11.3%–58.9%). All resistant isolates carrying β-lactamase genes also had plasmid replicons, mainly IncFII and IncFIB (41.7%–60.5%). These findings suggest that voluntary or mandatory prohibition of the off-label use of ceftiofur in breeder hatcheries is necessary to prevent the dissemination of ceftiofur-resistant E. coli throughout the stages of integrated broiler production.