Variations in blood thyroid hormone concentrations give an indirect indication of changes in thyroid gland activity; therefore, selecting a suitable method to determine these hormones is crucial. The aim of this study was to compare serum thyroid hormone concentrations (i.e. T4, T3, fT4 and fT3) using chemiluminescence immunoassay (CLIA) and enzyme-linked immunosorbent assay (ELISA) methods in broiler breeders. This study was carried out on 30 blood samples from randomly selected 45-week-old broiler breeders. Blood samples were collected in summer from the wing vein of birds by using disposable syringes. The separated serum was analysed to determine thyroxine (T4), triiodothyronine (T3), free thyroxine (fT4) and free triiodothyronine (fT3) concentrations using the mentioned methods. Data were processed in excel and statistical analysis was carried out using SPSS/ver17 software. Our data from this study indicates there was a significant difference in the T4 (P < 0.0001), fT4 (P < 0.01) and fT3 (P < 0.05) concentrations between the two methods and that the levels of these hormones were higher when using the CLIA method, except for fT3. The linear regression analysis of these hormones showed that the CLIA and ELISA results were significantly correlated only for fT4 (P < 0.0001, r = 0.798) concentration. In conclusion, determination of the thyroid hormones using the CLIA method had an acceptable performance of higher sensitivity and precision and was more appropriate than the ELISA method for clinical application and investigation in poultry.