ABSTRACT: Background: Radiomics has been widely used to non-invasively mine quantitative information from medical images and could potentially predict tumor phenotypes. Pathologic grade is considered a predictive prognostic factor for head and neck squamous cell carcinoma (HNSCC) patients. A preoperative histological assessment can be important in the clinical management of patients. We applied radiomics analysis to devise non-invasive biomarkers and accurately differentiate between well-differentiated (WD) and moderately differentiated (MD) and poorly differentiated (PD) HNSCC. Methods: This study involved 206 consecutive HNSCC patients (training cohort: n = 137; testing cohort: n = 69). In total, we extracted 670 radiomics features from contrast-enhanced computed tomography (CT) images. Radiomics signatures were constructed with a kernel principal component analysis (KPCA), random forest classifier and a variance-threshold (VT) selection. The associations between the radiomics signatures and HNSCC histological grades were investigated. A clinical model and combined model were also constructed. Areas under the receiver operating characteristic curves (AUCs) were applied to evaluate the performances of the three models. Results: In total, 670 features were selected by the KPCA and random forest methods from the CT images. The radiomics signatures had a good performance in discriminating between the two cohorts of HNSCC grades, with an AUC of 0.96 and an accuracy of 0.92. The specificity, accuracy, sensitivity, positive predictive value (PPV), and negative predictive value (NPV) of the abovementioned method with a VT selection for determining HNSCC grades were 0.83, 0.92, 0.96, 0.94, and 0.91, respectively; without VT, the corresponding results were 0.70, 0.83, 0.88, 0.80, and 0.84. The differences in accuracy, sensitivity and NPV were significant between these approaches (p < 0.05). The AUCs with VT and without VT were 0.96 and 0.89, respectively (p < 0.05). Compared to the combined model and the radiomics signatures, The clinical model had a worse performance, and the differences were significant (p < 0.05). The combined model had the best performance, but the difference between the combined model and the radiomics signature weren't significant (p > 0.05). Conclusions: The CT-based radiomics signature could discriminate between WD and MD and PD HNSCC and might serve as a biomarker for preoperative grading.