Numerous biomolecular markers have been studied to improve the accuracy of fine needle aspiration biopsy (FNAB) in the diagnostic and prognostic evaluation of thyroid tumors, but none of them has yet become clinically useful. The recently discovered BRAF mutation, which occurs specifically in papillary thyroid cancers (PTC) with a high prevalence and is associated with poor clinicopathological outcomes, has the potential to be a useful diagnostic and prognostic marker for PTC. In the present study, we investigated whether detection of BRAF mutation on FNAB specimens was technically possible and could be used as an adjunct diagnostic tool with routine FNAB. Evaluation of a new colorimetric mutation detection method demonstrated 100% sensitivity and 100% specificity in comparison with conventional DNA sequencing as the "gold standard" in a large pool of DNA samples from various primary thyroid tumor specimens and cell lines. We found this novel technique even more sensitive in detecting BRAF mutation on FNAB specimens than conventional sequencing. In a series of 48 patients undergoing thyroidectomy, mostly for thyroid cancer or for suspicion of cancer, we performed preoperative FNAB and, using the colorimetric mutation detection method, identified BRAF mutation on the cytological specimens. Prospective analysis showed that 50% of the nodules that proved to be PTC on surgical histopathology were correctly diagnosed by BRAF mutation analysis on FNAB specimens; there were no false positive findings. Thus, we have demonstrated the usefulness of BRAF mutation detection on FNAB specimens that can help diagnose and identify those PTC patients who may need more aggressive surgical treatment and vigilant clinical monitoring.