Background: Trismus, a restricted mouth opening in head and neck cancer patients may be caused by tumor infiltration in masticatory muscles, radiation-induced fibrosis or scarring after surgery. It may impede oral functioning severely. The aims of our study were to determine: (1) the incidence of trismus at various time points; and (2) the patient, tumor, and treatment characteristics that predict the development of trismus after radiotherapy in head and neck cancer patients using a large database (n ¼ 641). Methods: Maximal mouth opening was measured prior to and 6, 12, 18, 24, 36, and 48 months after radiotherapy. Patient, tumor, and treatment characteristics were analyzed as potential predictors for trismus using a multivariable logistic regression analysis. Results: At six months after radiotherapy, 28.1% of the patients without trismus prior to radiotherapy developed trismus for the first time. At subsequent time points the incidence declined. Over a total period of 48 months after radiotherapy, the incidence of trismus was 3.6 per 10 person years at risk. Patients who had tumors located in the oral cavity, oropharynx or nasopharynx, and the salivary glands or ear, and who had a longer overall treatment time of radiotherapy, were more likely to develop trismus in the first six months after radiotherapy. Maximal mouth opening was a predictor for developing trismus at all time points. Conclusion: Incidence of trismus is 3.6 per 10 person years at risk. Tumor localization and overall treatment time of radiotherapy are predictors for developing trismus the first six months after radiotherapy. Maximal mouth opening is a significant predictor for developing trismus at all time points. Regular measurements of maximal mouth opening are needed to predict trismus.