Introduction: Methadone is the most common opioid in use for opioid substitution therapy. The relation of methadone and electrocardiographic findings is nearly well known while the relationship between its electrocardiographic indexes and hormonal changes is not well recognized. Objective: To evaluate the hormonal changes in patients who are taking methadone maintenance treatment (MMT) and its effects on electrocardiographic indexes, in comparison with healthy control groups. Patients and Methods: 40 patients receiving MMT therapy for at least last six months and 40 healthy subjects were enrolled in the study. Serum estradiol, testosterone, luteinizing hormone, follicle stimulating hormone and thyroid function tests were measured. Mean QT Interval, P-R Interval (PRi) and QRS duration were also documented in maximum. Results: There were no significant differences in hormonal parameters between MMT and control groups. No significant relation was found between hormonal parameters, dose and duration of methadone usage in patients group. QTc was significantly higher in methadone users than control groups. QTc had a significant negative correlation with Testosterone level (r=-0.581, P= 0.007) in males. Significant difference was found between PRi in patients and control groups (P= 0.007). Conclusion: Electrocardiographic changes are an important complication of methadone that seems to be related to low testosterone level in men.