Mediastinal lymph node metastases (MLNM) from differentiated thyroid carcinoma (DTC) are considered difficult to diagnose. The aim of this study was to assess the value of iodine-131 (131I) single photon emission tomography/computed tomography (SPET/CT) and of 18F-fluorodeoxyglucose (18F-FDG) positron emission tomography/computed tomography (PET/CT) for the diagnosis of MLNM from DTC. Five hundred and eleven consecutive patients operated for DTC and treated with 131I for ablation of the remnant thyroid and/or for treatment of metastases were enrolled in the study and underwent an 131I whole body scan (131I-WBS). Thirty seven sites of increased 131I uptake, on the 131I-WBS that could be an indication for MLNM were re-evaluated by a 131I-SPET/CT scan. Thirty four other patients with negative 131I-WBS but having elevated serum thyroglobulin (Tg), were examined by 18F-FDG PET/CT to possibly diagnose MLNM. A total of 44 DTC patients with MLNM were identified, among the above 37 and 34 cases: 25/37 (67.6%) cases were examined and identified by 131I-SPET/CT and 19/34 (55.9%) cases by 18F-FDG PET/CT. A total of 25 and 19 cases were identified. The male-to-female ratio and the average age in patients with 18F-FDG-avid MLNM were significantly higher than in patients with 131I-avid MLNM. Among the above 44 patients, 40 patients had superior mediastinal nodal metastases, 9 had aortic nodal metastases and only 1 inferior mediastinal nodal metastases. A patient could have metastases in more than one site. In conclusion, our study suggests that in 511 operated DTC patients, treated for remnant ablation and/or for metastases and examined by 131I-WBS, there were 37 cases doubtful of having MLNM in the 131I-WBS and 34 cases doubtful, because of negative 131I-WBS and elevated Tg. The 131I-SPET/CT scan was sensitive for detecting MLNM in 25 of the 37 cases and the 18F-FDG PET/CT in 19 of the 34 cases. These hybrid imaging modalities, when applied as above, were suitable for detecting more MLNM and thus, better supporting treatment planning in these DTC patients.