Objective This study aims to explore the clinicopathologic features of patients with acquired immune deficiency syndrome (AIDS) with cytomegalovirus (CMV) infection, and to analyze the differential diagnosis with other common viral infections and non-infectious diseases. Methods A total of 38 patients confirmed CMV-positive by immunohistochemical staining, HIV-positive, with complete clinical data and meeting the inclusion criteria were collected from the Department of Pathology, Shanghai Public Health Clinical Center from 2013 to 2023. A comprehensive analysis was conducted based on histological morphology, special staining, in situ hybridization, and clinical data. Result The main symptoms of the patients were fever, nausea and vomiting, gastric discomfort, abdominal pain and diarrhea, while a few cases presented with hematemesis, hematochezia, cough, and hemoptysis. Tissue samples from 32 cases were obtained via gastrointestinal endoscopic biopsies, including 10 cases with biopsies from more than 2 sites. 2 cases were from lymph nodes, and 1 case each from skin, submandibular gland, lung, and perianal biopsies. Pathological results showed gastrointestinal mucosal erosion with acute and chronic inflammation, with ulceration forming in 3 cases. Lymph node biopsies showed reactive lymphoid follicle hyperplasia. Acute and chronic inflammatory cell infiltration with focal coagulative necrosis was observed in skin, perianal tissue, submandibular gland, and lung specimens. In this study, 35 patients showed enlargement of epithelial cells, endothelial cells, macrophages, and fibroblasts, with eosinophilic viral inclusions in the nuclei surrounded by a clear halo, resembling an "owl's eye". In the other 3 patients, no definite inclusion bodies were observed in their biopsy specimens (gastrointestinal mucosal tissue). Immunohistochemical testing showed that all 38 cases were positive for CMV and negative for herpes simplex virus (HSV) and latent membrane protein (LMP). Special staining revealed that acid-fast staining was positive for Mycobacterium in 3 cases, while periodic acid Schiff stain and methenamine silver staining detected one case each of Talaromyces marneffei in the transverse colon, Candida albicans in the stomach, and Aspergillus in the lungs. The results of in situ hybridization for Epstein-Barr encoded RNA (RNAEBER) were negative in all 38 cases. Conclusion The distinctive pathological feature of AIDS patients with CMV infection is the enlargement of infected cells, with eosinophilic viral inclusions in the nuclei resembling an "owl's eye". Combined with immunohistochemistry, precise locali-zation of CMV expression within the cells can be achieved. Additionally, special staining and in situ hybridization provide robust evidence for the differential diagnosis of lesions caused by EBV, HSV, respiratory viruses, and Hodgkin's lymphoma.