<jats:title>Abstract</jats:title><jats:sec><jats:title>Objective</jats:title><jats:p>Graves' disease (GD) is the most common cause of hyperthyroidism. In many cases, when the aetiological diagnosis of GD is not evident based on the clinical evaluation and thyroid function testing, it may become challenging to distinguish Graves' hyperthyroidism from other forms of thyrotoxicosis.</jats:p><jats:p>The current study was primarly carried out to compare the diagnostic effectiveness of two TSH receptor antibody immunoassays (IMAs), ultrasonography and thyroid scintigraphy in hyperthyroidism scenario.</jats:p></jats:sec><jats:sec><jats:title>Methods</jats:title><jats:p>We retrospectively analysed consecutive patients with newly diagnosed and untreated thyrotoxicosis who underwent thyroid functional tests, both TRAb and TSI measurements, thyroid scintigraphy and ultrasonography. TRAb assessment was carried out by Kryptor<jats:sup>®</jats:sup> compact PLUS, while TSI by Immulite<jats:sup>®</jats:sup>. Echo pattern 3 corresponded to ‘thyroid inferno’, and the final diagnosis of GD vs non‐Graves' hyperthyroidism was made according to the thyroid scan (qualitative scintigraphy). Receiver operating characteristic (ROC) curves were drawn using the final diagnosis as reference. Clinical sensitivity and specificity, accuracy, positive predictive value (PPV) and negative predictive value (NPV) were calculated for all the tests.</jats:p></jats:sec><jats:sec><jats:title>Results</jats:title><jats:p>A total of 124 untreated hyperthyroid patients were included in our study (GD, n = 86 vs non‐Graves' hyperthyroidism, n = 38). ROC curves showed that the optimal cut‐off values associated with the highest diagnostic sensitivity and specificity was 0.7 IU/L for TRAb Kryptor<jats:sup>®</jats:sup> (93 [85.4‐97.4] and 86.8 [71.9‐95.5]) and 0.1 IU/L for TSI Immulite<jats:sup>®</jats:sup> (94.2 [86.9‐98.1] and 84.2 [68.7‐93.9]), respectively. For the echo pattern 3, we found a good sensitivity (92.1%) and a high PPV (95.2%) but a quite low specificity value (69.8%) and a relative low NPV (57.5%). For thyroid scintigraphy, the TcTU cut‐off value of 1.3% corresponded to the best limit for sensitivity and specificity in our patients (95.3 [88.5‐98.7] and 96.4 [81.6‐99.4]). The Passing‐Bablok regression equation and the Bland‐Altman test showed a great degree of correlation and agreement existed between TRAb Kryptor<jats:sup>®</jats:sup> and Immulite<jats:sup>®</jats:sup> TSI results.</jats:p></jats:sec><jats:sec><jats:title>Conclusions</jats:title><jats:p>Thyroid scintigraphy remains the most accurate method to differentiate causes of thyrotoxicosis. However, TRAb assays can be alternatively adopted in this setting, limiting the use of thyroid scintigraphy (TcTU evaluation) to TRAb‐negative patients. Thyoid US is less accurate than both TRAb/TSI and thyroid scintigraphy, but the ‘thyroid inferno’ pattern provides a high PPV for GD.</jats:p></jats:sec>