Objective: To investigate the features of metastasis in the lymph nodes alongside of the internal jugular vein (IJV) in differentiated thyroid cancer (DTC) and the significance of biopsy of lymph nodes around the IJV during surgery. Methods: The clinical and pathological data of 98 DTC patients (106 cervical sides) undergoing biopsy of the lymph nodes alongside of the IJV in radical thyroidectomy from January 2006 to June 2012 were reviewed. Patients were divided into lymph node metastasis (cN1b) group and lymph node negative (cN0) group according to the preoperative physical and imaging findings, and the data between preoperative examinations and postoperative pathological results were compared. Results: Forty-six (43.4%) of the 106 cervical sides of the lymph nodes alongside of the IJV after biopsy were positive, which included 39 of the 56 sides in cN1b group and 7 of the 50 sides in cN0 group. So, the false positive and negative rates were 28.3% (17/60) and 15.2% (7/46), respectively. Conclusion: DTC frequently metastasizes to the lymph nodes alongside of the IJV. Decision based on preoperative examinations is insufficient to identify whether the lateral neck lymph nodes are involved, so the routine biopsy of lymph nodes alongside of the IJV during radical surgery for DTC is recommended.