This study aimed to determine the feasibility, diagnostic efficacy, and safety of ultrasound-guided core needle biopsy (CNB) as a first-line biopsy method for cervical lymphadenopathy of non-thyroid origin. This retrospective cohort study included consecutive patients with cervical lymphadenopathy in whom US-guided CNB was used as the first-line biopsy method for cervical lymph nodes (LNs) of presumed non-thyroid origin. The coaxial CNB technique was routinely used, while the tilting and hydrodissection CNB techniques were selectively employed for small high-risk LNs. The primary endpoint of this study was the diagnostic efficacy of CNB, evaluated by the rate of inconclusive results (nondiagnostic and indeterminate) and diagnostic accuracy (criterion 1: malignant results; criterion 2: malignant or indeterminate result). The secondary outcomes included the feasibility and safety of CNB, assessed based on the technical success rate and complication rate, respectively. The rates of nondiagnostic, indeterminate, and inconclusive results were 0.7%, 3.4%, and 4.1%, respectively. The sensitivity, specificity, and accuracy of CNB for malignant LNs were 96.2%, 100%, and 97.8%, respectively, with criterion 1, and these values were all 99.8% with criterion 2. The technical success rate of CNB was 99.3%. There were no major complications and 7 cases (0.6%) of minor complications (asymptomatic hematomas). CNB was technically feasible, effective, and safe as a first-line biopsy method for cervical lymphadenopathy of non-thyroid origin with high diagnostic accuracy for malignant nodal disease. Question The role of US-guided CNB as a first-line biopsy method for cervical LNs has not yet been verified and established. Findings US-guided CNB, as a first-line method, demonstrated a high technical success rate and diagnostic accuracy for malignant nodes, with few minor complications. Clinical relevance US-guided CNB can be used as an effective first-line biopsy method for cervical lymphadenopathy and will enable accurate diagnosis of malignant LNs.