Many cancer genes form mutation hotspots that disrupt their functional domains or active sites, leading to gain- or loss-of-function. We propose a mutation set enrichment analysis (MSEA) implemented by two novel methods,MSEA-clust and MSEA-domain, to predict cancer genes based on mutation hotspot patterns. MSEA methods are evaluated by both simulated and real cancer data. We find approximately 51% of the eligible known cancer genes form detectable mutation hotspots. Application of MSEA in eight cancers reveals a total of 82 genes with mutation hotspots,including well-studied cancer genes, known cancer genes re-found in new cancer types, and novel cancer genes.