Objectives-To evaluate the diagnostic performance of ultrasound (US)-guided fine-needle aspiration with optional core needle biopsy of head and neck lymph nodes and masses, with attention to differences between biopsy of treated squamous cell carcinoma (SCC) and biopsy of other lesions.Methods-Institutional Review Board approval was obtained, and the need for consent was waived for this retrospective study. All 861 US-guided biopsies of head and neck lymph nodes and masses performed between March 1, 2012, and May 16, 2016, were reviewed.Results-Of the 861 biopsies, 53 targeted SCC with residual masses after treatment. The biopsy procedures yielded benign or malignant pathologic results in 71.7% (38 of 53) of treated SCC and 90.7% (733 of 808) of all other lesions (P < .001). A reference standard based on subsequent pathologic results or clinical and imaging follow-up was established in 68.4% of procedures. In cases with benign or malignant biopsy results and a subsequent reference standard, the sensitivity values for malignancy were 87.5% (95% confidence interval, 64.0%-96.5%) in treated SCC and 98.3% (95% confidence interval, 96.0%-99.3%) in all other cases (P = .047), and the specificity values were 63.6% (95% confidence interval, 35.4%-84.8%) in treated SCC and 99.5% (95% confidence interval, 97.3%-99.9%) in all other cases (P < .001). There were no major complications related to the biopsy procedures.Conclusions-Excluding treated SCC, US-guided fine-needle aspiration with optional core needle biopsy of head and neck lymph nodes and masses has excellent diagnostic performance. Needle biopsy of head and neck SCC with a residual mass after therapy has a high rate of nondiagnostic samples, suboptimal sensitivity, and poor specificity.