Preoperative radiotherapy is a widely accepted treatment procedure in rectal cancer. Radiation-induced changes in the tumor are well described, whereas less attention has been given to the non-neoplastic mucosa. Our aim is to provide a detailed analysis of the morphological features present in non-neoplastic mucosa that pathologists need to be familiar with, in order to avoid misdiagnosis, when evaluating rectal cancer specimens of patients preoperatively treated with radiotherapy, especially with short-course regimen. We compared 2 groups of 95 rectal cancer patients treated preoperatively with either short-course (45 patients) or long-course radiotherapy (50 patients). Depending on the type of protocol, different histopathological features, in terms of inflammation, glandular abnormalities and endocrine differentiation were seen in the non-neoplastic mucosa within the irradiated volume. Of note, features mimicking dysplasia, such as crypt distortion, nuclear and cytoplasmic atypia of glandular epithelium, were identified only in the short-course group. DNA mutation analysis, using a panel of 56 genes frequently mutated in cancer, and p53 immunostaining were performed on both tumor and radiation-damaged mucosa in a subset of short course cases. Somatic mutations were identified only in tumors, supporting the concept that tissues with radiation-induced “dysplastic-like” features are not genetically transformed. Pathologists should be aware of the characteristic morphological changes induced by radiation. The presence of features simulating dysplasia in the group treated with short-course radiotherapy may lead to serious diagnostic mistakes, if erroneously interpreted. Next generation sequencing (NGS) analysis further validated the morphological concept that radiation-induced abnormalities do not represent pre-neoplastic lesions.