The aim of this prospective study (ClinicalTrials.gov: NCT01880203) was to evaluate the diagnostic and prognostic value of a 7-panel mutation testing in the aspirates of thyroid nodules with indeterminate cytology (IC). Methods Eligible patients had a thyroid nodule ≥15 mm with IC (Bethesda III-V) for which surgery had been recommended. Detection of BRAF and RAS mutations was performed using pyrosequencing and RET/PTC and PAX8/PPARγ rearrangements using Real-Time quantitative reverse transcription-polymerase chain reaction (RT-PCR). Results Among 131 nodules with IC, 21 (16%) were malignant including 20 differentiated cancers and one thyroid lymphoma. Molecular abnormalities were identified in 15 nodules with IC corresponding to 10 malignant and 5 benign tumours. BRAF mutation was detected in 4 nodules all corresponding to classic PTC, and PAX8/PPARγ rearrangement in 2 HCC. In contrast, RAS mutation was identified in eight nodules, of which four were malignant, and one RET/PTC3 rearrangement in a follicular adenoma. This data resulted in an accuracy of 88%, sensitivity of 48%, specificity of 95%, positive-predictive value of 67%, and negative-predictive value of 91%. After a 56 month's follow-up, the proportion of excellent response was similar in patients with molecular alterations (67%) and those without (60%). Conclusions By increasing the overall risk of cancer from 16 to 67% in mutated nodules and by diminishing it to 9% in wildtype, this study confirms the relevance of the 7-panel mutation testing in the diagnostic of nodules with IC. Genetic testing, however, did not predict outcome in the cancer patient subgroup.