Minhua Ye1 *,* Sijia Ren2 *,* Chunguo Wang,1 Xiaoshun Shi,3 Jianfei Shen1 1Department of Cardiothoracic Surgery, Taizhou Hospital of Zhejiang Province Affiliated to Wenzhou Medical University, Wenzhou, 317000, People’s Republic of China; 2Department of Cardiothoracic Surgery, Taizhou Hospital of Zhejiang Province Affiliated to Zhejiang University School of Medicine, Hangzhou, 310000, People’s Republic of China; 3Department of Thoracic Surgery, Nafang Hospital, Southern Medical University, Guangzhou, 510515, People’s Republic of China*These authors contributed equally to this workCorrespondence: Jianfei ShenDepartment of Cardiothoracic Surgery, Taizhou Hospital of Zhejiang Province Affiliated to Wenzhou Medical University, Wenzhou, 317000, People’s Republic of ChinaEmail jianfei051@163.comXiaoshun ShiDepartment of Thoracic Surgery, Nanfang Hospital, Southern Medical University, No. 1838 North Guangzhou Avenue, Guangzhou, 510515, People’s Republic of ChinaEmail sxs917@163.comBackground: Primary adenoid cystic carcinoma (ACC) of the lung, which arises from the bronchial gland and is rare, accounting for only 0.04– 0.2% of all primary lung tumors. The genetic profiling of bilateral ACC of unknown primary site and application in postoperative decision-making are less reported.Case Presentation: A 57-year-old male with a smoking history of over 30 years and multiple nodules in both lungs was present to our department. After assessing the bilateral solid nodules in his Positron Emission Tomography–Computed Tomography (PET/CT) scan, malignant lesions at the left lower lung, right lower lung, and right middle lung are suspected. Sequential selective video-assisted thoracoscopic surgeries (VATS) were performed. A genetic alteration test of 425 cancer-related genes and global gene expression profile of the specimens revealed intrapulmonary metastasis existed. The patient was followed up for three years without recurrence and tissue mutations in liquid biopsy.Conclusion: We present a way of omics-based multiple pulmonary lesions origin assessment, facilitating post-operative differential diagnosis and treatment decision for difficult cases.Keywords: adenoid cystic carcinoma, next-generation sequencing, lung cancers of unknown primary site, PIK3R1