Background: Cytokeratin (CK) intermediate filaments are demonstrated to have enormous potential in regulating cellular motility and cancer progression. There are more than 20 divergent CKs that have been identified, of which CK 8, 17, 18 and 19 are reported to be elevated in the tumour biopsies of head and neck cancer squamous cell carcinoma (HNSCC) patients. However, CK expression profiles in the saliva of HNSCC patients have not been investigated. We aim to investigate the mRNA expression profiles of CKs in saliva collected from healthy controls, HPV-negative and -positive HNSCC patients. Methods: Oral rinse samples were collected from 42 cancer-free healthy controls (age-matched) and patients who have been diagnosed with HPV-negative (n = 20) and -positive (n = 48) HNSCC. Results: Here, we report that the mRNA expression profiles of CKs differed in saliva collected from healthy controls and HNSCC patients. The mRNA expression levels of CK 8 and 18 were significantly elevated in saliva collected from HPV-negative HNSCC patients; whilst, CK 17 and 19 were expressed at a higher mRNA level in saliva collected from HPV-positive HNSCC patients compared to healthy controls. Importantly, receiver operating characteristic (ROC) analysis showed salivary CK 8 and 18 to have superior sensitivity and specificity in discriminating the HPV-negative HNSCC patients from healthy controls (80% and 86%) as well as between HPVnegative and -positive HNSCC patients (75% and 81%). Conclusion: In summary, we have demonstrated that an aberrant expression of salivary CKs may serve as a potential non-invasive diagnostic biomarker in HNSCC.