Background: Laying hens, which are widely utilized for consumption and export in various regions, experience prolonged antibiotic exposure due to their longer lifespan, increasing the risk of antibiotic resistance and impacting the microbial environment of poultry slaughterhouses. Given the significance of extended-spectrum-β-lactamase (ESBL)-producing Escherichia coli in food safety, this study aimed to investigate the prevalence of ESBL genes in E. coli isolated from a laying hen slaughterhouse in Konya, Turkey. Methods: Sampling was conducted using a convenient sampling approach, and a total of 150 samples were collected from a single slaughterhouse over six visits during both warm (June-August) and cold (January-March) seasons to evaluate seasonal variations. Samples were categorized into environmental sources (personnel, air, wastewater, eggs) and carcass-related sources (cloaca, carcasses at critical control points, final product). Classical cultural and molecular techniques and antimicrobial susceptibility tests were used for ESBL presence and gene characterization. For sequence analysis, the bidirectional Sanger Gene sequence analysis method was applied. Results: PCR-based detection identified 10 of the 17 isolates as E. coli by amplifying the uspA gene, and bidirectional Sanger sequencing further confirmed these isolates at the species level. The E. coli isolates were detected at various sampling areas, including personnel, carcasses after evisceration, and raw wastewater samples collected at different time points. In the multiplex PCR analysis, most ESBL isolates were positive for the blaCTX-M gene. The co-existence of blaTEM and blaCTX-M genes was detected in five samples. Additionally, three genes (blaSHV, blaCTX-M, and blaOXA) were identified in a carcass sample after evisceration. All ESBL-producing isolates harbored the blaCTX-M1 gene, and multiple antibiotic resistance was observed across all isolates. The presence of these genes was strongly associated with resistance to ampicillin, amoxicillin-clavulanic acid, aztreonam, cefepime, cefpodoxime, cefuroxime, and cephalothin, highlighting the critical role of blaCTX-M in driving the multidrug resistance patterns observed in this study. The highest resistance rate (80%) was observed in "personnel" and "carcass samples after evisceration", while all isolates remained sensitive to carbapenems (imipenem and meropenem). Conclusions: Our findings highlight the importance of the laying hen slaughter line as a potential source of contamination with ESBL-producing E. coli, which poses significant implications for food safety and public health. These findings underscore the need for improved control measures to mitigate ESBL E. coli transmission in poultry processing and highlight the importance of optimizing antibiotic use strategies in laying hen farming.