MicroRNA (miRNA) microarray analysis has consistently found altered expression of miRNAs in thyroid tumors, suggesting their roles in thyroid carcinogenesis. To explore whether this differential expression can be used as a diagnostic tool in surgical pathology and fine-needle aspirate (FNA) specimens, the expression of selected miRNA was evaluated by quantitative RT-PCR, using total RNA from 84 formalin-fixed paraffin-embedded tissues and 40 ex vivo aspirate specimens. miRNA from all paraffin-embedded tissues and all but one FNA sample were found to be analyzable, with paraffin sections yielding better miRNA quality. Preliminary analysis of 6 miRNAs in 10 papillary thyroid carcinoma and 10 follicular adenoma identified significant overexpression of miR-146b, -221, and -222 in papillary thyroid carcinoma (P<0.02), but not miR-146a, -155, or -187 (P>0.08). The expression of these first three miRNAs was examined in a series of 5 normal thyroid, 11 hyperplastic nodules, 24 follicular adenoma, 27 classical papillary thyroid carcinoma, 5 follicular variant papillary thyroid carcinoma, 2 follicular carcinoma, and 10 encapsulated follicular lesions with partial nuclear features of papillary carcinoma. Results showed miR-146b to be most consistently overexpressed in both classical papillary carcinoma and follicular variants, whereas all other groups showed lower expression at a similar level (P<0.001 for pair-wise comparisons between papillary carcinoma and all other groups). Follicular lesions with partial features of papillary carcinoma all showed low miR-146b levels similar to other non-papillary carcinoma groups, suggesting that they are biologically distinctive from papillary carcinoma. miR-221 and miR-222 also showed higher expression in papillary carcinoma, but with substantial overlaps with the other groups. When applied to 40 FNA samples of various lesions, only miR-146b and miR-222 persisted as distinguishing markers for papillary carcinoma. We concluded that miRNAs, particularly miR-146b, might potentially be adjunct markers for diagnosing papillary thyroid carcinoma in both FNA and surgical pathology specimens.