PURPOSEImmune checkpoint blockade (ICB) therapy has significantly improved clinical outcomes in bladder cancer. Identification of correlates of benefit is critical to select appropriate therapy for individual patients.METHODSTo reveal genetic variables associated with benefit from ICB, we performed whole-exome sequencing on tumor specimens from 88 patients with advanced bladder cancer treated with ICB.RESULTSWe identified several genetic factors that correlated with progression-free and overall survival after ICB therapy including ARID1A mutation, tumor mutational burden, intratumoral heterogeneity, the ratio of nonsynonymous to synonymous mutations in the immunopeptidome (immune dN/dS), and tumor cell purity. In addition, we noted that neutrophil-to-lymphocyte ratio and smoking history were negatively associated with overall survival. These genetic characteristics define four molecular subtypes demonstrating differential sensitivity to ICB. We validated the association of these four subtypes with clinical benefit from ICB in an independent cohort (IMvigor210). Finally, we showed that these molecular subtypes also correlate with outcome, although with distinct relationships, among patients not treated with ICB using The Cancer Genome Atlas (TCGA) bladder cancer cohort. Using parallel RNA sequencing data, the subtypes were also shown to correlate with immune infiltration and inflammation, respectively, in the IMvigor210 and TCGA cohorts.CONCLUSIONTogether, our study defines molecular subgroups of bladder cancer that influence benefit from ICB.