Thyroid carcinoma (TC) accounts for ~2.1% of newly diagnosed cancer cases. Mutations in KRAS, HRAS, NRAS and BRAF are primary participants in the development and progression of various types of malignancy, including differentiated TC (DTC). Therefore, the present prospective cohort study aimed to screen patients with DTC for variations in RAS gene family and BRAF gene. Exon 1 and 2 of KRAS, HRAS, NRAS and exon 15 of BRAF gene were screened for hotspot mutations in 72 thyroid tumor and adjacent normal tissue samples using di-deoxy Sanger sequencing. HRAS T81C mutation was found in 21% (15 of 72) of DTC tissue samples, therefore this mutation was investigated in blood samples from patients with DTC and controls as a genetic polymorphism. In addition, HRAS T81C genotypes were determined in 180 patients with DTC and 220 healthy controls by performing restriction fragment length polymorphism. BRAFV600E mutation was confined to classical variant of papillary thyoid cancer (CPTC; 44.4%) and was significantly associated with multifocality and lymph node (LN) metastasis. No mutation was found in exons 1 and 2 of KRAS and NRAS and exon 2 of HRAS genes, however, mutation was detected in exon 1 of HRAS gene (codon 27) at nucleotide position 81 in 21% (15 of 72) of DTC tumor tissue samples. Furthermore, HRAS T81C single nucleotide polymorphism was significantly associated with the risk of DTC with variant genotypes more frequently detected in cases compared with controls (P≤0.05). Moreover, frequency of variant genotypes (TC+CC) was significantly higher among DTC cases with no history of smoking, males, greater age, multifocality and LN metatasis compared with healthy controls (P<0.05). BRAFV600E mutation was primarily present in CPTC and associated with an aggressive tumor phenotype but mutations in RAS gene family were not present in patients with DTC. HRAS T81C polymorphism may be involved in the etiopathogenesis of DTC in a Pakistani cohort. Furthermore, testing for the BRAFV600E mutation may be useful for selecting initial therapy and follow-up monitoring. [ABSTRACT FROM AUTHOR]