Abstract Context: Ultrasound (US)-guided fine-needle aspiration biopsy (FNAB) is the most reliable nonsurgical test for distinguishing benign from malignant thyroid nodules. However, there is no consensus on which nodules should undergo FNAB. Aims: The aims of this study were to evaluate the utility of US-guided FNAB in the diagnostic assessment of nodules with or without clinical/US features suggestive for malignancy and to investigate the additional contribution of BRAF V600E mutation analysis in the detection of differentiated thyroid cancer. Design and Methods: Thyroid cytoaspirates from 2421 nodules at least 4 mm in diameter were performed in 1856 patients who underwent cytological evaluation and biomolecular analysis. Results: Cytology showed high positive predictive value and specificity for the diagnosis of malignant lesions. BRAF V600E mutation was found in 115 samples, 80 of which were also cytologically diagnosed as papillary thyroid cancer. BRAF mutation analysis significantly enhanced the diagnostic value of cytology, increasing FNAB diagnostic sensitivity for malignant nodules by approximately 28%. Micro PTC (63% of diagnosed papillary thyroid carcinoma) showed a high prevalence of multifocality, extrathyroidal extension, and lymph node metastases, underlining the malignant potential of thyroid microcarcinomas. Each investigated US/clinical characteristic of suspected malignancy correlated with the presence of a thyroid cancer in thyroid nodules with diameter of at least 4 mm. Conclusions: These data indicate that nodules of at least 4 mm may underlie a thyroid cancer independently of US/clinical characteristics of suspected malignancy, suggesting the need to perform FNAB. The diagnostic sensitivity for thyroid cancer is significantly increased by BRAF V600E mutation analysis, indicating that the screening for BRAF mutation in FNAB samples has a relevant diagnostic potential.