Hatcheries have the power to spread antimicrobial resistant (AMR) pathogens through the poultry value chain because of their central position in the poultry production chain. Currently, no information is available about the presence of AMR Escherichia coli strains and the antibiotic resistance genes (ARGs) they harbor within hatchezries. Therefore, this study aimed to investigate the possible involvement of hatcheries in harboring hemolytic AMR E. coli. Serotyping of the 65 isolated hemolytic E. coli revealed 15 serotypes with the ability to produce moderate biofilms, and shared susceptibility to cephradine and fosfomycin and resistance to spectinomycin. The most common β-lactam resistance gene was blaTEM, followed by blaOXA-1, blaMOX-like, blaCIT-like, blaSHV and blaFOX. Hierarchical clustering of E. coli isolates based on their phenotypic and genotypic profiles revealed separation of the majority of isolates from hatchlings and the hatchery environments, suggesting that hatchling and environmental isolates may have different origins. The high frequency of β-lactam resistance genes in AMR E. coli from chick hatchlings indicates that hatcheries may be a reservoir of AMR E. coli and can be a major contributor to the increased environmental burden of ARGs posing an eminent threat to poultry and human health.