Abstract : The present study has been designed to investigate hyperthyroidism on thyroid receptor gene expression levels in hypothalamic and pituitary tissues in prepubertal and pubertal male rats. The present study has been conducted in the animal house of the college of veterinary medicine, AL-Qadisiya University during the period extended from Feb-July,2022. Thirty-two premature (aged 40 days) and thirty-two mature (aged 70 days) male rats. Divided to 4 groups each 16 rats 2 subgroups 10 and 20 days as following, Pre-pubertal control group (C-pre): 16 premature male rats (aged 40 days) will be orally administered for 20 days with distilled water. Pre-pubertal hyperthyroid group (H-pre): 16 premature male rats (aged 40 days) will be orally administered for 20 days with thyroxine (T4) in drinking water (0.002% w/v) beside intragastric gavage of 200 T4 μg/kg body weight. Post-pubertal control group (C-pre): 16 mature male rats (aged 70 days) will be orally administered for 20 days with distilled water. Premature hyperthyroid group (H-pre): 16 mature male rats (aged 70 days) will be orally administered for 20 days with thyroxine (T4) in drinking water (0.002% w/v) beside intragastric gavage of 200 T4 μg/kg body weight. At the end of each treated and control subgroup period, males were anaesthesized (by injection of 0.3ml ketamine + 0.1 ml of xylazine/ kg b.w. ip), dissected and blood samples were obtained from abdominal vein in non-heparinized tubes. Hypothalamic, testicular and pituitary sample from each male has been obtained for evaluation of mRNA expression levels of GAPDH as housekeeping gene and TR-α and TR-β genes using qRT-PCR technique based on Syber Green dye. Hypothalamic, testicular and pituitary samples obtained from all groups recorded significant higher RNA concentrations at 10 and 20 day periods compared with control. Significant elevation of both TR-α and TR-β genes expression levels (fold changes) have been shown in thyroxine treated males in comparison with control, started after 10 days of treatment and continued in its elevation after 20 days of treatment. In comparison between period, both genes showed a significant difference between periods of control group, whereas thyroxine treated group recorded significant gradual elevation of both genes as the treatment period progress at 10th and 20th days.