Currently, no definitive diagnostic tool is available to distinguish unifocal and multifocal papillary thyroid carcinoma (PTC). This study aims to identify potential diagnostic markers of multifocal PTC. In 471 Hashimoto's thyroiditis (HT) patients, the significant difference was revealed in anti-thyroid peroxidase antibody (TPOAb) concentration, the cytokeratin-19 (CK-19) expression, the occurrence of the B-Raf proto-oncogene serine/threonine kinase (BRAF) mutations and the rearrangement in transformation (RET)/PTC. The patients' samples were assayed for the expression of CK-19, cyclooxygenase-2 (COX-2), galectin-3, and the protein human bone marrow endothelial cell marker-1 (HBME-1) using immunohistochemistry. The BRAF gene mutation was detected using a sequencer. Differences were examined using the Kruskal-Wallis test and the Chi-squared and Fisher's exact tests. The results showed that the elevated CK-19 expression, and the presence of BRAF mutations and RET/PTC rearrangements were indicators of multifocal PTC in HT, suggesting the need for total bilateral thyroidectomy. Among HT patients with TPOAb > 1300 IU/Ml, the occurrence of central lymph node metastasis is significantly higher in multi-focal PTC than single-focal PTC. Therefore, these markers may prove useful for discerning between uni- and multifocal PTC, thereby preventing unnecessary surgery in the treatment of unifocal PTC and promoting sufficient treatment of multifocal PTC.