Background: In the past decade, systematic and comprehensive analyses of cancer genomes have identified cancer driver genes and revealed unprecedented insight into the molecular mechanisms underlying the initiation and progression of cancer. These studies illustrate that although every cancer has a unique genetic make-up, there are only a limited number of mechanisms that shape the mutational landscapes of cancer genomes, as reflected by characteristic computationally-derived mutational signatures. Importantly, the molecular mechanisms underlying specific signatures can now be dissected and coupled to treatment strategies. Systematic characterization of mutational signatures in a cancer patient's genome may thus be a promising new tool for molecular tumor diagnosis and classification. Results: In this review, we describe the status of mutational signature analysis in cancer genomes and discuss the opportunities and relevance, as well as future challenges, for further implementation of mutational signatures in clinical tumor diagnostics and therapy guidance. Conclusions: Scientific studies have illustrated the potential of mutational signature analysis in cancer research. As such, we believe that the implementation of mutational signature analysis within the diagnostic workflow will improve cancer diagnosis in the future.