Background: Proper candidates to improve the effectiveness of molecular testing for thyroid nodules detected on ultrasonography (US) in a clinical setting are not well known. We aimed at evaluating the effective indication and method of BRAFV600E mutation analysis of aspiration specimens according to the US features of thyroid nodules in a BRAFV600E mutation-prevalent area. Methods: A total of 244 patients with 244 thyroid nodules were prospectively classified as malignant and nonmalignant based on US. Thyroid nodules with any malignant US features including spiculated margin, the presence of microcalcifications or macrocalcifications, marked hypoechogenicity, or a taller-than-wide shape were defined as US-positives and those without these features were defined as US-negatives. All patients underwent US-guided fine-needle aspiration (FNA). The presence of the BRAF V600E mutation in FNA specimens was determined by allele-specific polymerase chain reaction (AS-PCR) and direct DNA sequencing. The mutation results were correlated with cytology and either surgical pathology or follow-up. Results: Of 244 nodules, 66 were US-positive and 178 were US-negative. The malignancy rate was 92% (61/66) for US-positives and 14% (25/178) for US-negatives. The BRAFV600E mutation was identified in 67% (44/66) of US-positives and in 10% (17/178) of US-negatives. The BRAF V600E mutation for nodules with indeterminate or nondiagnostic cytology was present in 45% (5/11) of US-positives and in 8% (2/26) of US-negatives ( p=0.0168). A false negative cytology with the mutation was found in only one case of the US-negatives. All nodules with the mutation were surgically confirmed as papillary carcinomas. Adding the genetic analysis to the FNA as compared with the FNA alone improved the sensitivity and accuracy for US-positives, whereas there was no significant improvement for US-negatives. With regard to sensitivity and accuracy, the use of the AS-PCR was better than the use of the direct DNA sequencing for US-positives as compared with US-negatives. Conclusion: The application of BRAFV600E mutation analysis in FNA specimens is more effective for thyroid nodules with malignant US features as compared with nodules without malignant US features. The use of the AS-PCR is more valuable as compared with the direct DNA sequencing to refine the diagnosis in a clinical setting.