Abstract: Recently, the incidence of papillary thyroid carcinoma (PTC) has been increasing yearly. Ultrasonography is considered the first tool in the screening and diagnosis of thyroid nodules. Ultrasound-guided fine needle aspiration (US-FNA) biopsy is an effective, minimally invasive intervention for the differential diagnosis of thyroid nodules before operation. The diagnosis of US-FNA is classified by the Bethesda diagnostic system. Although fine needle aspiration cytology has high sensitivity and specificity, unclear diagnoses, such as atypical follicular lesions, are still existed. US-FNA combined with genetic mutation analysis (BRAF, RAS, and RET/PTC rearrangement) for such atypical lesions (Bethesda Ⅲ-Ⅴ), as well as microRNA analysis, can significantly improve the diagnostic accuracy of indefinite nodules. This process facilitates standardized clinical management of benign and malignant thyroid nodules.