This study's objective was to evaluate if electrocardiogram (ECG) parameters differ depending on euthanasia method, and if welfare implications result. Sixteen mature laying hens previously outfitted with subcutaneous wireless telemetry and housed in conventional cages or aviaries were used. Manual cervical dislocation (CD) and 30% CO2 displacement were chosen. Continuous ECG data collection began 5 min prior to the procedure to establish a baseline, then continued during and following euthanasia until complete cessation of cardiac activity. Insensibility and euthanasia were determined by an experienced handler to be loss of posture and the end of neuromuscular convulsions with no noticeable breathing. The attending veterinarian monitored and confirmed successful euthanasia via auscultation of the heart. Veterinary confirmation occurred within 5 min for all hens, thus a 5-min sampling period was used. Samples of ten successive QRS complexes per hen per minute were used to calculate average QRS complex amplitude and heart rate. Data were analyzed using the Glimmix Procedure in SAS 9.4, and reported as deviations from baseline. Housing was not a significant factor (p > 0.05). Heart rate showed a significant effect for method (p = 0.0232), time (p < 0.0001), and method*time interaction (p = 0.0001). Compared to baseline, heart rate for CD was 238 bpm higher at minute 1 (p < 0.0001) and 106 bpm higher at minute 2 (p = 0.0027) compared to CO2. While there was no significant effect of method on QRS amplitude (p = 0.6220), there was a time effect (p = 0.0266). Cervical dislocation as a method of euthanasia may induce a greater stress response in laying hens compared to CO2 displacement.