The aim of this study was to evaluate the frequency (as qualitative analysis) and level (as quantitative analysis) of promoter hypermethylation of four genes, P16, TSHR, RASSF1A and RARβ2, and to assess their diagnostic or prognostic values in papillary thyroid tumors. Fifty formalin-fixed paraffin-embedded (FFPE) samples consisting of 25 malignant tumors and 25 non-malignant thyroid tumors were analyzed using COBRA method. Promoter hypermethylation of P16, TESH, RASSF1A and RARB2 genes was noted not only in 10, 7, 19 and 13 cases of malignant tumors, but also it was detected in 7, 11, 23 and 8 cases of benign tumors, respectively, limiting its diagnostic usefulness. The quantitative hypermethylation level was significantly higher in malignant tumors compared to benign tumors for P16 (P<0.004), TSHR (P<0.006) and RASSF1A (P<0.001), but the methylation level of RARβ2 (P<0.31) showed considerable overlap between the two groups. The mean levels of hypermethylation of P16, TSHR and RASSF1A genes were significantly higher in malignant papillary thyroid tumors compared to benign tumors and by choosing the appropriate cutoff for each gene, we could distinguish 9, 9 and 8 PTCs from 25 cases by P16, RASSF1A and TSHR methylation analysis, respectively. According to our results, these three genes, in combination, may be useful as molecular markers. The findings of present study imply that the P16, TSHR and RASSF1A gene promoter hypermethylation may play important roles in the pathogenesis of PTC and can be a potential biomarker for selecting patients with PTC.