BACKGROUND: Antimicrobial resistance (AMR) has been deepening in the layer poultry sector in Zambia partly due to the inappropriate use of antimicrobials. Escherichia coli (E. coli), a commensal and zoonotic bacterium, can potentially be a source of AMR. OBJECTIVES: This study assessed the phenotypic AMR profiles of E. coli isolated from the apparent health-laying hens in Lusaka and Copperbelt provinces of Zambia. METHODS: A cross-sectional study was conducted between September 2020 and April 2021 in which 365 cloacal swabs were collected from 77-layer farms based in Lusaka and Copperbelt provinces of Zambia. E. coli isolation and identification were done using cultural and biochemical properties and confirmed using the 16S rRNA gene sequencing. Antimicrobial susceptibility testing (AST) was done using the Kirby-Bauer disc-diffusion method. Data analysis was done using WHONET 2020 and Stata v.16.1. RESULTS: Of the 365 samples, E. coli was isolated from 92.9% (n = 339). The AMR was detected in 96.5% (n = 327) of the isolates, of which 64.6% (n = 219) were multidrug-resistant (MDR). E. coli was highly resistant to tetracycline (54.6%) and ampicillin (54%) but showed low resistance to meropenem (0.9%), ceftazidime (6.2%) and chloramphenicol (8.8%). CONCLUSION: This study found a high prevalence of E. coli resistant to some commonly used antibiotics in poultry, which is a public health concern because of the potential contamination of eggs and layers of chicken meat that enter the food chain. Urgent attention is needed, including strengthening antimicrobial stewardship and surveillance programmes in layer poultry production in Zambia.