Tumor stem cells (TSC), which are considered as likely candidates for the origin of cancer, are deduced to be responsible for tumor metastasis theoretically. We therefore investigated whether TSC were associated with lymph node metastasis in supraglottic carcinoma. Immunohistochemistry was performed for CD44, CD133, and LYVE-1 to detect TSC and lymphatic vessel density (LVD) in 66 primary supraglottic carcinoma tissue samples from 30 patients with lymph node metastasis (N+) and 36 patients without (N0). Reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot were used to detect the expression of CD44 and CD133 at mRNA and protein levels in N+ and N0 primary tumors. The LVD was 22.4 ± 10.26 in 30N+ and 6.8 ± 4.09 in 36N0 samples subjected to immunohistochemistry, which was associated with their clinical nodal stages. There were 43.33% CD44-positive and 93.33% CD133-positive samples in 30N +, and 13.89% CD44-positive and 44.44% CD133-positive samples in 36N0 (P < 0.05). However, in each positive slide, there were only 5∼10% CD44-positive cells, but 70∼85% CD133-possitive cells. The expressions of CD44 and CD133 of N+ obtained through RT-PCR and Western blot were significantly higher than those of N0. These results suggest that TSC identified through CD44-positive cells in N+ were significantly higher than those in N0, indicating that TSC may be responsible for lymph node metastasis. CD133, whose expression is not restricted to TSC, may be unspecific for TSC identification in hypostatic supraglottic carcinoma.