Context: Fine-needle aspiration cytology (FNAC) is the gold standard for the differential diagnosis of thyroid nodules but has the limitation of inadequate sampling or indeterminate lesions. Objective: We aimed to verify whether search of thyroid cancer-associated protooncogene mutations in cytological samples may improve the diagnostic accuracy of FNAC. Study Design: One hundred seventy-four consecutive patients undergoing thyroid surgery were submitted to FNAC (on 235 thyroid nodules) that was used for cytology and molecular analysis of BRAF, RAS, RET, TRK, and PPRγ mutations. At surgery these nodules were sampled to perform the same molecular testing. Results: Mutations were found in 67 of 235 (28.5%) cytological samples. Of the 67 mutated samples, 23 (34.3%) were mutated by RAS, 33 (49.3%) by BRAF, and 11 (16.4%) by RET/PTC. In 88.2% of the cases, the mutation was confirmed in tissue sample. The presence of mutations at cytology was associated with cancer 91.1% of the times and follicular adenoma 8.9% of the time. BRAF or RET/PTC mutations were always associated with cancer, whereas RAS mutations were mainly associated with cancer (74%) but also follicular adenoma (26%). The diagnostic performance of molecular analysis was superior to that of traditional cytology, with better sensitivity and specificity, and the combination of the two techniques further contributed to improve the total accuracy (93.2%), compared with molecular analysis (90.2%) or traditional cytology (83.0%). Conclusions: Our findings demonstrate that molecular analysis of cytological specimens is feasible and that its results in combination with cytology improves the diagnostic performance of traditional cytology. Search of a complete panel of oncogene mutations in cytological specimens of thyroid nodules is feasible and improves the diagnostic performance of traditional cytology.