Fine-needle aspiration biopsy (FNAB) samples from thyroid tumor tissues were analyzed for the presence of the BRAF(T1796A) mutation by polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) analysis. This assay utilized a specific mismatched primer and has proved to be a relatively simple, accurate, and highly sensitive method. The analysis of 130 aspirated samples from thyroid tumors (18 follicular adenomas, 72 papillary carcinomas [PTCs], 8 follicular carcinomas, 2 undifferentiated carcinomas, 1 medullary carcinoma, 2 malignant lymphomas, and 27 adenomatous goiters) revealed BRAF(T1796A) mutations in 37 (51.4%) of 72 PTCs, supporting the usefulness of this method. We examined BRAF(T1796A) in 21 patients with thyroid tumors using leftover cells in the needle at the preoperative FNAB. BRAF(T1796A) was detected in 4 patients, of which 3 cases were diagnosed as positive and 1 case as suspicious by cytologic examination. Furthermore, BRAF(T1796A) mutations were found to occur more often in tumors of 3 cm or larger in size. Our results indicate that the preoperative determination of the presence of a BRAF(T1796A) mutation by conventional PCR-RFLP may be potentially useful in the diagnosis of the most common thyroid malignancies.