Purpose Our aim is to determine the impact of the size of ROI in detecting subcentimeter metastatic lymph nodes in patients with head and neck squamous cell carcinoma (HNSCC). Secondary aim is to determine the impact of the histopathologic grade of the primary cancer on the ADC value of the metastatic nodes. Methods The prospective study included 51 patients with histopathologically proven HNSCC at the primary site. Primary site includes oral cavity, oropharynx, larynx, and hypopharynx. ADC values of the lymph nodes were measured on ADC maps by placing two ROIs in the lymph nodes (0.2 cm 2 in the center of the node and the whole node). Lymph nodes were dissected by levels, marked by the surgeon, and sent to the pathologist. Results By applying a smaller ROI, ADC values have greater sensitivity, specificity, NPV, PPV, and AUC in detecting metastasis compared to the ADC value of the entire node (88.0%, 80.73%, 90.7%, 75.9%, 0.912% versus 80.0%, 77.98%, 85% ,71.4%, and 0.819%, respectively) p < 0.001. Statistically significant negative correlation was established between the tumor grade and the ADC of lymph node at ROI 0.2 cm 2 and ROI of the whole lymph node (rho = − 0.425; p = 0.002, and rho = − 0.298; p = 0.038, respectively). Conclusion ROI size affects the ADC value of the nodes. The higher histopathological grade of the primary tumor is inversely correlated with the ADC value of the lymph nodes