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Background: BRAFV600E, the most common gene mutation, is detected in approximately 50% of sporadic papillary thyroid carcinoma (PTC) and may be associated with triggering tumorigenesis of PTC. The aim of our study was to discover additional mutations to increase the diagnostic performance of molecular tests in screening for thyroid cancer from fine needle aspiration biopsy (FNAB) specimens. Methods: DNA was extracted from 120 freshly obtained FNAB specimens selected according to cytopathology grades of the Bethesda system. A conventional BRAFV600E test was carried out with real-time PCR, and further mutation screening for mutations in codons 464, 466, BRAF 469, NRAS KRAS codons 12/13 and 61 was done by, pyrosequencing. Histopathology reports were reviewed for those who underwent thyroidectomy (n=83). Results: The real-time PCR method detected 45 BRAFV600E-positive cases whereas pyrosequencing detected 30 cases. Additional BRAF (n=4), NRAS (n=11) and KRAS (n=3) mutations were detected in 17 cases (one overlapping BRAF and NRAS mutation). Among 11 NRAS-mutated cases, eight were confirmed as PTC and one as FVPTC on histopathology reports. Five PTC-confirmed cases with BRAFV600E mutation showed additional mutations, all of which were mutations. Discussion: Despite the higher sensitivity of real-time PCR for detecting BRAFV600E mutations, pyrosequencing easily detected additional point mutations. NRAS mutations were the most prevalently identified additional mutations and were highly associated with malignancy. In conclusion, our findings demonstrate that additional mutations identified by pyrosequencing may help in the pre-operative process in determining the possibility of malignancy and further studies on the occurrence of simultaneous mutations of and may be warranted. [ABSTRACT FROM AUTHOR]