BACKGROUND Chordoma is a rare malignant tumor of fetal notochord origin that occurs along the spinal axis. The fine-needle aspiration biopsy (FNAB) findings are described, correlated with histology and radiology, and compared with previously reported descriptions of chordoma. METHODS Fine-needle aspiration biopsies of 12 cases of chordoma with histologic confirmation were reviewed. Imaging studies were reviewed in seven cases. Cytologic material included smears, ThinPrep, and cell blocks. Immunostains were performed on selected cytologic and histologic specimens. Multiple cytologic parameters were studied. RESULTS Eleven specimens were from the spinal axis, and one was from a chest wall metastasis. Ten cases were diagnosed as chordoma on cytologic material, one was positive for malignancy with a differential diagnosis of chordoma and well differentiated chondrosarcoma, and one was positive for malignancy, not further classified. Most smears were moderately to highly cellular and demonstrated typical physaliphorous cells and a myxoid background. Two of the 10 cases diagnosed as chordoma showed pleomorphic physaliphorous cells, nuclear inclusions, and binucleation. Nuclear inclusions were observed in three other cases diagnosed as chordoma. Histologic follow-up of one case with pleomorphic physaliphorous cells showed conventional chordoma with focal areas of increased cellularity and pleomorphism. Pleomorphic sarcomatous cells were the predominant cell type in one case that showed dedifferentiated chordoma histologically. Mitotic figures were rarely observed in cytologic material. CONCLUSIONS Cytomorphologic features of chordoma allow accurate diagnosis by FNAB. Features associated with dedifferentiation include increased pleomorphism of physaliphorous cells and may include nuclear inclusions, bi- or multinucleation, and rarely, mitotic figures. Cancer (Cancer Cytopathol)