Fine-needle aspiration (FNA) is commonly used as a preoperative assessment to diagnose thyroid cancer. However, misdiagnosis of malignancy by FNA is not rare, even if image examination suggests the possibility of thyroid cancer. In the present study, the clinicopathological factors of patients whose preoperative FNA examination had not led to a diagnosis of thyroid cancer were examined. In total, 125 patients with thyroid cancer who underwent FNA and surgery (total thyroidectomy, subtotal thyroidectomy or hemithyroidectomy) at the Department of Surgery, Surgical Oncology and Science of the Sapporo Medical University Hospital between 2006 and 2013 were retrospectively analyzed. The patients were divided into two groups: Group A, malignancy determined by FNA, and group B, no malignancy. The groups were then compared by gender, age, tumor size, stage, tumor stage, lymph node metastasis, histology, surgical procedure methods, presence or absence of calcification and thyroglobulin levels. The mean age of the patients in group A (5 males and 59 females) was 53.0 years. The mean age in group B (11 males and 49 females) was 54.2 years. The mean tumor size in both groups was 1.6 cm. The mean thyroglobulin levels were 82.7 ng/ml in Group A and 525.5 ng/ml in group B. There were also significant differences between the groups for tumor stage (P=0.046), histological type (P=0.024) and thyroglobulin levels (P=0.035). The results of the present study suggested that it may be difficult to diagnose thyroid cancer by FNA in cases with non-papillary carcinoma and higher thyroglobulin levels.