Objective: To analyze the relationship between clinical characteristics and pathogenic gene mutations in multifocal papillary thyroid carcinoma (MPTC) and to investigate the proportion of independent primary tumors (IP) versus intrathyroidal metastases (ITM) in MPTC. Additionally, to explore the correlation between specific gene mutations and clinical features such as multifocality, tumor size, and lymph node metastasis. Methods: Patients with multifocal thyroid tumor meeting inclusion criteria were consecutively enrolled. Two lesions per case were selected for preoperative ultrasound-guided fine-needle aspiration biopsy (FNAB). All lesions were pathologically confirmed as papillary thyroid carcinoma postoperatively. FNAB samples were subjected to multi-gene panel testing and classified into three groups based on mutational profiles of 26 thyroid-related genes: (1) identical mutations in both lesions (intrathyroidal metastasis), (2) completely discordant mutations (independent primary tumors), and (3) shared mutations with an additional mutation in one lesion (uncertain origin). Results: Among 58 initially enrolled MPTC patients, 8 were excluded due to noncompliant specimens. The final cohort included 50 patients (37 females, 13 males) with a mean age of 42.64 +/- 11.12 years. The median tumor diameter was 12.5 (IQR: 7.6, 20.0) mm, with 38.0% (19/50) classified as papillary thyroid microcarcinoma. A total of 128 mutations, 4 gene fusions, and 3 gene amplifications were detected across 100 qualified FNAB samples. BRAF V600E was the most prevalent mutation (84.0%, 84/100), followed by DICER1 (7.0%), PTEN (6.0%), and RET (4.0%). Identical mutational profiles (intrathyroidal metastasis) were observed in 64.0% (32/50) of cases, while 18.0% (9/50) exhibited completely discordant mutations (independent primary tumor). The remaining 18.0% (9/50) showed shared mutations with an additional mutation, predominantly in smaller lesions. The incidence of completely different mutations in ipsilateral lesions and bilateral lesions was different (p = 0.030). No significant correlation between BRAF V600E and clinical characteristics such as tumor size, multifocality, capsular invasion or lymph node metastasis (p > 0.05). Conclusion: Multi-gene panel testing of preoperative FNAB samples effectively discriminates clonal relationships in MPTC, revealing distinct molecular profiles between ITM and IP. The high prevalence of BRAF V600E mutations and frequent clonal homogeneity underscore the necessity of comprehensive genetic profiling to guide personalized management. Routine multi-lesion sampling is advocated for optimizing risk stratification and surgical decision-making.