Abstract: Cancer of unknown primary (CUP) is a rare malignant tumor with a poor prognosis. Searching for the primary lesion, analyzing tissue type or molecular immune characteristics are of great significance to improve the prognosis of in patients with CUP. In the era of precision medicine, next-generation sequencing (NGS) can be used on tumor tissue or peripheral blood samples to detect CUP at the genetic level and subsequently guide diagnosis and treatment, prolong patient survival, and improve prognosis. This review discusses NGS sample selection, tissue traceability, molecular characteristics of CUP, and NGS-guided treatment of CUP.