Background and Aims Fine-needle aspiration cytology (FNA) has limitations due to the small sample size obtained, including nondiagnostic specimens, indeterminate cytological results, and false-negative or false-positive results, potentially leading to misdiagnosis or missed diagnoses. The BRAF gene mutation, specifically BRAF^V600E, is a specific biomarker for papillary thyroid carcinoma (PTC). However, studies on its diagnostic value in FNA benign high-risk thyroid nodules are limited. This study was conducted to further explore the clinical value of adding BRAF^V600E mutation testing in FNA benign thyroid nodules.Methods A retrospective analysis was conducted on the clinical data of 549 patients who underwent 668 ultrasound evaluations indicating high risk of PTC and were classified as TIRADS categories 4-5 thyroid nodules at the Thyroid Surgery Department of China-Japan Union Hospital of Jilin University from January 2019 to September 2022. All patients underwent surgical treatment, and paraffin pathological examination was performed on resected tissues after surgery. Based on inclusion and exclusion criteria, 84 FNA benign thyroid nodules were included in this study. The clinicopathologic characteristics of nodules with BRAF^V600E mutations were analyzed. Using postoperative pathology as the gold standard, the diagnostic performance of BRAF^V600E mutation testing in FNA benign nodules was assessed.Results Among the 84 FNA benign thyroid nodules, 44 (52.4%) tested positive for the BRAF^V600E mutation. Patients with BRAF^V600E-positive nodules were more likely to be younger than 45 years (56.8% vs. 35.0%, P=0.045), and their nodules had a smaller median long diameter compared to the BRAF^V600E-negative group (0.49 cm vs. 0.61 cm, P=0.024). Postoperative pathology revealed 63 PTC nodules and 21 benign nodules. PTC nodules had a smaller median long diameter than benign nodules (0.50 cm vs. 0.70 cm, P=0.004) and a higher proportion of nodules <1 cm (95.2% vs. 71.4%, P=0.007), with a higher BRAF^V600E mutation rate (68.3% vs. 4.8%, P<0.001). In terms of the ultrasound characteristics of thyroid nodules, BRAF^V600E-positive nodules showed a significantly higher rate of blurred/irregular margins than the negative group (86.4% vs. 60.0%, P=0.006). Similarly, PTC nodules showed a higher rate of blurred/irregular margins compared to benign nodules (81.0% vs. 52.4%, P=0.010). Multivariate Logistic regression analysis indicated that BRAF^V600E-positive thyroid nodules had a 39.184-fold higher risk of being diagnosed as PTC compared to BRAF^V600E-negative nodules (P=0.001), with BRAF^V600E mutation being an independent risk factor for PTC diagnosis. The sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of BRAF^V600E mutation testing for PTC diagnosis were 69.3%, 95.2%, 97.7%, 50.0%, and 75.0%, respectively.Conclusion BRAF^V600E mutation testing demonstrates high positive predictive value and accuracy, and can reduce the risk of missed PTC diagnoses among FNA-reported benign thyroid nodules. It is recommended that thyroid nodules with highly suspicious ultrasound features and TIRADS categories 4-5 undergo combined FNA and BRAF^V600E mutation testing.