Objectives: The aim of this study was to investigate the antimicrobial resistance (AMR) patterns of Escherichia coli strains isolated from poultry flocks suffering from colibacillosis in Greece and to detect the presence of the mcr-1 gene in isolates being phenotypically resistant to colistin. Results: A total of 150 E. coli strains were isolated from commercial layers and layer breeder flocks in Greece and tested for antimicrobial susceptibility. A high level of susceptibility was revealed for cephalosporins, neomycin, and colistin. Susceptibility varied for other antimicrobials (tetracycline, doxycycline, lincospectin, trimethoprim/sulfamethoxazole, enrofloxacin, amoxicillin), whereas no susceptibility was reported for macrolides, tiamulin, lincomycin, oxacillin. Concerning colistin resistance, 20 E. coli strains were found to be phenotypically resistant (13 strains showed intermediate resistance pattern and 7 strains fully resistance trait). Further investigation was performed by PCR, which has revealed the presence of the mcr-1 gene in one phenotypically colistin-resistant isolate. Conclusion: AMR is prevalent in layer poultry production, including resistance against colistin confirmed by the presence of the mcr-1 gene.