Abstract: The development of high-throughput sequencing technology has significantly advanced genomic research. Somatic mutations in the cancer genome are caused by various mutational processes, each of which may produce mutational signature. These mutational signatures provide novel insights into the etiology of cancer and reveal endogenous and exogenous factors that influence cancer onset and progression. Mutational signatures are increasingly shown to be closely related to clinical diagnosis and treatment, and can thus be used as biomarkers of cancer and indicators for predicting treatment efficacy and prognosis. In this review, we comprehensively discuss the association between mutational signatures and tumor etiology, molecular subtyping, therapeutic prediction, prognostic evaluation, and tumor origins. This review aims to provide insightful references for clinical diagnosis and treatment.