Differentiated-type Intraepithelial Neoplasia (DIN) is defined as HPV-negative squamous intraepithelial proliferation with abnormal keratinocyte differentiation and basal cell atypia, originally described in the vulva, with following descriptions in the oral cavity. DIN occurring in the anus is quite rare, and to the best of our knowledge, only one publication reported it. In this report, we describe the clinicopathological features of this entity on anal margin, associated with invasive squamous cell carcinoma. In addition, using the next generation sequencing (NGS) technique, we have demonstrated TP53 mutation in the invasive component but not in the associated DIN-like lesion, where p53 immunohistochemical expression was restricted to basal layers.