Thyroglossal duct cyst (TDC), or embryologic remnants of thyroid gland, is a common congenital anomaly. TDC may cause a midline neck mass, which occasionally may become infected, and rarely gives rise to carcinoma. As a number of other nonneoplastic and neoplastic lesions can cause cystic masses in the neck, we explored the role of fine-needle aspiration (FNA) in making a preoperative diagnosis of TDC for a more accurate and timely clinical intervention. Twenty-six cases of TDC were identified from the cytopathology files of The Johns Hopkins Hospital in a 15-yr period (1990-2004). Material was obtained by FNA with or without radiologic (ultrasound) guidance. Smears were air-dried and stained with Diff-Quik, or wet-fixed and stained with Papanicolaou stain. Cytomorphologic characteristics were serially analyzed. Follow-up (tissue resection [n = 9] and clinical charts [n = 17]) was reviewed in all cases. Patients ranged in age from 8 to 83 yr (mean age, 55) with M:F ratio of 1.4:1. The size of the cyst ranged from 1.2 to 5 cm (mean 2.5 cm), as evaluated on radiological scans. The most common clinical presentation was a non-tender, mobile neck mass, which was painful on swallowing. Follow-up confirmed TDC in 18/26 cases (69%), whereas 8/26 cases resulted in various other benign lesions. During the same time period, 11/18 (61%) cases of surgically resected TDC were missed on prior FNA. Therefore, FNA showed a diagnostic sensitivity of 62% and a positive predictive value (PPV) of 69% for the diagnosis of TDC. The cytomorphologic features of TDC included the following: colloid (thick and fragmented, thin and watery, or mucinous), macrophages, lymphocytes, or predominantly neutrophils. The epithelium was ciliated columnar, metaplastic squamous or of mature squamous type. Thyroid epithelium was only rarely present (11%). FNA is only moderately sensitive for a preoperative evaluation of TDC. Cytomorphologic features are not unique; however, in the right clinicoradiologic setting should lead to an accurate diagnosis. Abundant colloid, most often with ciliated columnar epithelium, is the predominant cytopathologic finding. Thyroid epithelium is rarely identified. Differential diagnosis involves branchial cleft cyst, lymphoepithelial cyst, thyroid gland lesions, and lymphadenopathy (of various etiologies).