Several putative biomarkers have been reported to identify cancer stem cells (CSCs) in head and neck squamous cell carcinoma (HNSCC). Herein, we aimed to demonstrate the validity and the underlying relationship for these biomarkers in HNSCC. Bioinformatic analyses for the reported CSC biomarkers of HNSCC were performed based on the TCGA primary HNSCC cohort using the UCSC Xena browser. Targeted strategies for the validated biomarkers were searched and summarized. A total of 27 reported CSC biomarkers for HNSCC were identified and comprehensively evaluated. In regards to the expression pattern of CD44 in HNSCC, the expression patterns for the remaining 26 biomarkers presented 3 different tendencies. We managed to include all the 27 CSC biomarkers for HNSCC into 3 groups. Moreover, the biomarkers in each group indicated distinct clinicopathological features and a different overall survival status for HNSCC patients. The above information suggested the existence of CSC subpopulations in HNSCC. Accordingly, we demonstrated that precisely targeted strategies based on the CSC subgrouping clusters might effectively supplement conventional therapies, and benefit HNSCC patients. Further relevant studies are still necessary to improve treatment strategies for HNSCC based on the CSC area.